hey people,im really sorry for the super late posting..for this posting i will share with u guys the quality assurance and quality control in the histopathological laboratory where im attached to.
Quality Assurance(QA) and Quality Control(QC)
QA is a program in which overall activities conducted by the institution are directed towards assuring the quality of the products and services provided. QC is under QA in which products and services provided are evaluated in hopes of achieving high accuracy and reliability of test results and also aids troubleshooting. This involves continuous monitoring of operations and systemic day-today checking of the produced data to decide whether these are reliable enough to be released.
Application of QC protocol to:
Tissue processing
The ATPs are checked against maintenance checklist to ensure that all reagents are properly loaded and in sufficient amounts prior to start of processing. Reagents are maintained (after 20 cycles) to ensure a certain standard of quality of processing in subsequent cases.
Embedding
Paraffin dispensers are emptied monthly to clear debris collected at the bottom of the container.
Microtomy
The temperature of floatation baths are checked and recorded daily on the temperature log as part of equipment maintenance to ensure that it is in top condition during production of results. If out of range (less than 44oC or more than 50oC), corrective actions are taken by adjusting control knob. The microtomes and floatation baths are cleaned daily to remove debris after all sectioning done.
Staining
Working thermometer and equipment temperature display are calibrated monthly to make sure that readings displayed on ovens and refrigerators are within control range. The coldroom used to store special staining kits is inspected daily to ensure that it can function properly. The accuracy of its temperature is checked daily as the activity of special stains will be very much affected by temperature. When preparing staining reagents, the electronic balance may be used to measure out certain ingredients. Therefore, it is calibrated yearly to make sure the reading is accurate and within 1% of the reference mass as certain special stains are sensitive to quantity. Control samples are stained simultaneously with patients’ samples and observed for its quality. This is to ensure that any poor staining caused by defective reagents detected by control samples can be corrected at first hand before the sections are stained. H&E and special stains controls are evaluated and graded according to their quality from 5 (excellent) to 2 and below (inadequate in which remedial actions are to be taken). For H&E control, the nuclear and cytoplasmic control must be well defined and contrasted. As for special stains controls, there must be relatively good demonstration of 75% or more of the cells without background staining. During staining of patients’ samples, 10% of the slides contained in a rack are sent for microscopic examination for grading as part of QA. The slides are evaluated to detect any faults in staining, sectioning and mounting. Percentage of unsatisfactory slides caused by errors in staining, sectioning or mounting are counted monthly as well. Weekly, at the end of every Tuesday and Friday, reagents used for routine H&E are changed as part of QC so that the quality of staining can be consistent throughout. Records are maintained which indicate the type of reagent that is changed, filtered or replaced. Since Haematoxylin and eosin are prepared once a week, they are filtered daily to remove debris left by the previous slides. Before sending the slides for mounting, the machine is primed to remove any air bubbles present as it can affect the microscopic examination and thus, the quality of staining.
Dispatch slides
All slides are checked against tissue blocks prior to their dispatching. If unsatisfactory, they are
sent for recuts. Also, the number of slides indicated on request forms must tally against the amount being stained.
Lastly, pathologists evaluate the results in areas of sectioning, staining and mounting as these are the criteria that will affect the quality of section and thus, hinder diagnosis. The total number of errors that are being raised in all stages of workflow (from processing to labeling) are counted monthly and systematically documented in an ‘EVENTS’ file. In addition, a maintenance program for all equipment and instruments, listing the model and serial number, dates of services performed, parts installed, and the interval and date of the next service are kept and recorded. There is also a complete equipment inventory, listing the name and manufacturer of the instrument, model and serial number, purchased from, personnel who installed the instrument, the date, length of warranty, purchase price , and location of the manual of instructions covering the equipment.
This shall be the end of my posting. thanks for reading. :)
Sharon Ang
0503219H
Tuesday, November 6, 2007
Sunday, October 28, 2007
Week 18 Attachment Sharing
Alright, this week i'll be sharing more on the analysis, excision and identification of proteins. Also, i'll touch a bit on the DRP group that me and Jiaxin were put in charge of. This weeks blog is more of a reflective entry rather than a factual one.
After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.
Protein spot analysis
After extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.
For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.
Protein spot excision
The protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.
After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.
After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.
DRP students
For our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.
In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.
Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.
Johanna
0503309G
TG02
After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.
Protein spot analysis
After extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.
For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.
Protein spot excision
The protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.
After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.
After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.
DRP students
For our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.
In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.
Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.
Johanna
0503309G
TG02
Saturday, October 20, 2007
Week 17 Attachment Sharing
heya.......first and foremost i would like to wish all muslim frenz SELAMAT HARI RAYA.......dun forget to collect as many green packets as possible..it might be our last chance...hehehe....aniwei this week i would post something that was shown to me on the 1st day of work......ISOLATION OF PLASMA AND WBC..yah i noe..a bit outdated....
Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....
This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......
Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....
A) Isolation of plasma
1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min
-this separates the plasma from the other blood components
2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.
-pipette as much supernatant as possible but do not disturb the pellet
3. Store the eppendorf tubes at -80oC fridge
**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)
B) Isolation of WBC
1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes
-the pellet is actually RBC
2. Add 9ml of RBC lysis solution to the tubes
-this forms 1:3 blood to lysis solution ratio
3. Incubate the tubes at room temperature for 10 min
4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min
5. Pour away the supernatant
6. Add 1ml of RBC lysis solution and pipette up and down
7. Transfer everything from the tubes to eppendorf tubes
8.Centrifuge the tubes at 3000rpm for 3min at 4oC
9.Centrifuge at 15000rpm for 5 min at 4oC
Discard the supernatant
11. Store the pellet (WBC) at -80oC fridge
**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....
tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D
nur zahirah tg02
Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....
This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......
Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....
A) Isolation of plasma
1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min
-this separates the plasma from the other blood components
2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.
-pipette as much supernatant as possible but do not disturb the pellet
3. Store the eppendorf tubes at -80oC fridge
**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)
B) Isolation of WBC
1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes
-the pellet is actually RBC
2. Add 9ml of RBC lysis solution to the tubes
-this forms 1:3 blood to lysis solution ratio
3. Incubate the tubes at room temperature for 10 min
4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min
5. Pour away the supernatant
6. Add 1ml of RBC lysis solution and pipette up and down
7. Transfer everything from the tubes to eppendorf tubes
8.Centrifuge the tubes at 3000rpm for 3min at 4oC
9.Centrifuge at 15000rpm for 5 min at 4oC
Discard the supernatant
11. Store the pellet (WBC) at -80oC fridge
**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....
tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D
nur zahirah tg02
Wednesday, October 17, 2007
Week 16th-SPSS!!
Gomennasai, for the late posting!
Here's my posting for week 16th. Basically, for the past few weeks I have been involved in the statistics analysis for both of my gene projects using the SPSS software. SPSS is the abbreviation used for Statistical Package for the Social Sciences; it provides almost everything and anything you required to perform an analysis.
Common Statistics Perform in SPSS
In SPSS, it comprises of multiple statistics tests, most of them are covered in year 1 math stats module. Two of the more commonly features used are the descriptive statistics and the bivariate/multivariate statistics.
Descriptive statistics
The most fundamental and frequent statistic feature used. It summarized the samples’ results and portrays the tabulated results in forms of graphs or tables. Thence it is mainly used for quick and basic analysis. Here are some of the statistics tests used in descriptive statistics:
Frequencies
A simple measurement used to computes/determine the mean, median, modes and SD of the results (variables).
Cross tabulation
A cross tab provide information on 2 or more variables’ distribution consecutively and is usually presents a table format. Therefore, a cross tab is different from a frequency test. Some of the statistics tests used abide by with cross tab are chi-square (which test for clinical significant in variables), contingency coefficient (which test for strong interaction between variables) and phi coefficient (which test for degree of interaction between variables).
Bivariate/Multivariate Statistics
As the name implied, this feature is only suitable for 2 variables. Any statistics involving more variables would be under the multivariate statistics. Similarly here is a list of the some of the statistics tests used in bivariate statistics:
t-test
Commonly used when the sample size is minute or when the standard deviation is unknown. It is used to test if a null hypothesis is true or false (accept or reject). For example, if null hypothesis used is: mean of A = mean of B (where both the means of the variables is equal). After a series of calculation if t < a =" mean">
ANOVA
An ANOVA test compares the variables’ mean with their variances. Unlike t-test, ANOVA used variances instead of t value and also assess on more than two variables together.
So how and wat statistics should be applied to different data analysis?
The answer would depend on wat type of “story “ you wish to express in your research. For instance, if you wish to find out the relationship of drug X on blood glucose level. Then a linear regression approach would be an ideal.
A linear regression is a technique used to determine the relationship of dependent variables with independent variables. In this case, blood glucose level is your dependent variable while the dosage of drug X would be the independent variables.
Once you identify your variables you can start analyzed using both the descriptive statistics and the bivariate/multivariate statistics.
Another technique used for data analysis is called the factors analysis. This approach look into dependent variables, meanwhile, indirectly identifies the independent variables within. For example, by looking at blood glucose level (dependent variables), other independent variable such as concentration of drug X would be determined.
Other technique includes K-means cluster analysis, Hierarchical cluster analysis and Ordinal regression.
Lost in this statistics info??
Don’t worry maybe these definitions can help out.
Dependent variables
A dependent variable is an outcome, a prediction that can be influenced by the independent variable. Usually is something, which cannot be control.
Independent variables
An independent variable is something, which you may control or predict in experiment.
Means
Mean equal average
Null hypothesis
A null hypothesis is a presumed statement before statistics analysis
Data analysis
For SPSS to analysis a data/ samples results, a syntax is often used. Simply type in the code (instructions) you want the SPSS to do and select run. Subsequently, the results will appear in the output file in a graph, table, histogram formats (etc). Based on the tabulated results, interpretations can be made.
That all for this week!! Stay tune to next week SIP sharing!!
If you have any question on my post feel free to leave comment.
Avery (0503292E)
TG02
Here's my posting for week 16th. Basically, for the past few weeks I have been involved in the statistics analysis for both of my gene projects using the SPSS software. SPSS is the abbreviation used for Statistical Package for the Social Sciences; it provides almost everything and anything you required to perform an analysis.Common Statistics Perform in SPSS
In SPSS, it comprises of multiple statistics tests, most of them are covered in year 1 math stats module. Two of the more commonly features used are the descriptive statistics and the bivariate/multivariate statistics.
Descriptive statistics
The most fundamental and frequent statistic feature used. It summarized the samples’ results and portrays the tabulated results in forms of graphs or tables. Thence it is mainly used for quick and basic analysis. Here are some of the statistics tests used in descriptive statistics:
Frequencies
A simple measurement used to computes/determine the mean, median, modes and SD of the results (variables).
Cross tabulation
A cross tab provide information on 2 or more variables’ distribution consecutively and is usually presents a table format. Therefore, a cross tab is different from a frequency test. Some of the statistics tests used abide by with cross tab are chi-square (which test for clinical significant in variables), contingency coefficient (which test for strong interaction between variables) and phi coefficient (which test for degree of interaction between variables).
Bivariate/Multivariate Statistics
As the name implied, this feature is only suitable for 2 variables. Any statistics involving more variables would be under the multivariate statistics. Similarly here is a list of the some of the statistics tests used in bivariate statistics:
t-test
Commonly used when the sample size is minute or when the standard deviation is unknown. It is used to test if a null hypothesis is true or false (accept or reject). For example, if null hypothesis used is: mean of A = mean of B (where both the means of the variables is equal). After a series of calculation if t < a =" mean">
ANOVA
An ANOVA test compares the variables’ mean with their variances. Unlike t-test, ANOVA used variances instead of t value and also assess on more than two variables together.
So how and wat statistics should be applied to different data analysis?
The answer would depend on wat type of “story “ you wish to express in your research. For instance, if you wish to find out the relationship of drug X on blood glucose level. Then a linear regression approach would be an ideal.
A linear regression is a technique used to determine the relationship of dependent variables with independent variables. In this case, blood glucose level is your dependent variable while the dosage of drug X would be the independent variables.
Once you identify your variables you can start analyzed using both the descriptive statistics and the bivariate/multivariate statistics.
Another technique used for data analysis is called the factors analysis. This approach look into dependent variables, meanwhile, indirectly identifies the independent variables within. For example, by looking at blood glucose level (dependent variables), other independent variable such as concentration of drug X would be determined.
Other technique includes K-means cluster analysis, Hierarchical cluster analysis and Ordinal regression.
Lost in this statistics info??
Don’t worry maybe these definitions can help out.Dependent variables
A dependent variable is an outcome, a prediction that can be influenced by the independent variable. Usually is something, which cannot be control.
Independent variables
An independent variable is something, which you may control or predict in experiment.
Means
Mean equal average
Null hypothesis
A null hypothesis is a presumed statement before statistics analysis
Data analysis
For SPSS to analysis a data/ samples results, a syntax is often used. Simply type in the code (instructions) you want the SPSS to do and select run. Subsequently, the results will appear in the output file in a graph, table, histogram formats (etc). Based on the tabulated results, interpretations can be made.
That all for this week!! Stay tune to next week SIP sharing!!
If you have any question on my post feel free to leave comment.
Avery (0503292E)
TG02
Monday, October 1, 2007
Week 15!
Hello guys. My turn again! =) After 2 entries about protein expression (Immunohistochemistry/Immunofluorescence), this week, I will focus on GENE expression.
Anyway, I am currently extracting RNA from Formalin-fixed, Paraffin-embedded blocks. Hence, I shall talk about this process today yup!
Reason for extraction/isolation of RNA : Using this RNA extracted from the blocks, I will carry out Reverse-Transcription PCR to reverse-transcribe the mRNA I have gotten into cDNA and then carry out Real-time PCR to look at the genetic expression of a particular gene I am interested in. For more info on Real-time PCR, do refer to my group member, Avery’s blog entry (July 22, 07) as I believe she had written a very detailed entry about this technique =))
RNA isolation must be carried out over 2 days.
DAY 1 :
(Sectioning)
Using the microtome, I will section about 45um of tissue sections into a eppendorf tube.
Anyway, I am currently extracting RNA from Formalin-fixed, Paraffin-embedded blocks. Hence, I shall talk about this process today yup!
Reason for extraction/isolation of RNA : Using this RNA extracted from the blocks, I will carry out Reverse-Transcription PCR to reverse-transcribe the mRNA I have gotten into cDNA and then carry out Real-time PCR to look at the genetic expression of a particular gene I am interested in. For more info on Real-time PCR, do refer to my group member, Avery’s blog entry (July 22, 07) as I believe she had written a very detailed entry about this technique =))
RNA isolation must be carried out over 2 days.
DAY 1 :
(Sectioning)
Using the microtome, I will section about 45um of tissue sections into a eppendorf tube.
(Deparaffinissation)
Add 800uL of xylene into the tube and mix.
Significance: Since I am using FFPE blocks, I will first have to remove the wax from the section .
Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.
Add 800uL of Abs Ethanol into the tube and mix.
Significance: To remove the xylene from the sections completely. It is also the start of rehydration.
Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.
Add 800uL of 70% Ethanol into the tube, mix and repeat centrifuge.
Blot the tube briefly onto a paper towel to get rid of the ethanol residues.
Dry the tissue pellet for 10mins at 55ºC
Add 800uL of xylene into the tube and mix.
Significance: Since I am using FFPE blocks, I will first have to remove the wax from the section .
Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.
Add 800uL of Abs Ethanol into the tube and mix.
Significance: To remove the xylene from the sections completely. It is also the start of rehydration.
Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.
Add 800uL of 70% Ethanol into the tube, mix and repeat centrifuge.
Blot the tube briefly onto a paper towel to get rid of the ethanol residues.
Dry the tissue pellet for 10mins at 55ºC
(Overnight Proteinase K Digestion Incubation)
Add 100uL of Tissue Lysis Buffer, 16uL 10% SDS and 40uL proteinase K into the dried tubes and vortex briefly before incubating the tube overnight at 55ºC.
Significance: This is to disrupt the protein structures in order to get the mRNA we want
DAY 2:
(RNA isolation)
Add 325uL of binding buffer and abs ethanol to the tube and pipette to mix.
Add 100uL of Tissue Lysis Buffer, 16uL 10% SDS and 40uL proteinase K into the dried tubes and vortex briefly before incubating the tube overnight at 55ºC.
Significance: This is to disrupt the protein structures in order to get the mRNA we want
DAY 2:
(RNA isolation)
Add 325uL of binding buffer and abs ethanol to the tube and pipette to mix.
Significance: Binding buffer will bind to the nucleic acids we want and allow the contaminants to flow through the filter and into the collection for discard.
Combine the filter tube and collection tube and pipette the lysate into the upper reservoir
Centrifuge for 30 secs at 8000rpm and discard the flowthrough.
Add 500uL of Wash Buffer I to the upper reservoir. Centrifuge for 15secs @ 8000rpm. Discard flowthrough.
Combine the filter tube and collection tube and pipette the lysate into the upper reservoir
Centrifuge for 30 secs at 8000rpm and discard the flowthrough.
Add 500uL of Wash Buffer I to the upper reservoir. Centrifuge for 15secs @ 8000rpm. Discard flowthrough.
Add 500uL of Wash Buffer II and repeat centrifuge.
Add 300uL of Wash Buffer II and repeat centrifuge.
Significance: The nucleic acids will bound to the chaotropic salts specifically to the surface of the glass fibers pre-packed in the filter tube while all other contaminants (salts, proteins and other cellular contaminants) will be washed off the column.
Centrifuge the High Pure filter for 2 mins at max speed.
Place the High Pure filter tube into a fresh 1.5ml reaction tube, add 90uL Elution Buffer and centrifuge for 1 min @ 8000rpm before collecting the flowthrough.
Add 10uL DNase Incubation Buffer and 1.0uL DNase I to the eluate and mix. Incubate for 45mins at 37 ºC
Significance: DNase I will help to remove any residual DNA since all we want is the RNA from the blocks.
Add 20uL Tissue Lysis Buffer, 18uL 10% SDS and 40uL Proteinase K to the eluate. Vortex briefly and incubate for 1 hour at 55ºC
Significance: A second incubation step with Proteinase K is to improve on the quality of the RNA as well as to ensure that all protein structures are disrupted.
Repeat the steps involved for RNA ISOLATION until the part in which the High Pure filter is centrifuged at max speed for 2 mins.
This time round, add only 50uL of Elution Buffer and centrifuge for 1 min@ 8000rpm.
Using the NanoDrop spectrophotometer, we can then check the RNA concentration of the 50uL eluate we obtained from our RNA extraction! =)
All the reagents/buffer were from this kit known as High Pure RNA Paraffin Kit from Roche, Switzerland. Therefore, i do not know the content of some of the reagents such as the Wash Buffer I and II which is why i cannot specifically tell you guys what each and every one of the reagents does ya. Hope this is alright with you guys. But i have tried to explain as many steps as possible so the whole idea behind this RNA isolation process should be pretty clear =)
Difficulties Met During RNA extraction from Formalin-fixed Paraffin-embedded blocks:
(1) During sectioning, the tissue blocks are constantly exposed to the existence of RNase in the surrounding. Hence, the RNA in these blocks might risk getting destroyed during the sectioning process and affecting the total concentration of the RNA after the RNA extraction is done by the end of Day 2.
(2) The process of fixing the tissue sample and embedding it in paraffin has caused severe degradation of the RNA. Therefore, it’s more difficult to isolate good quality RNA from FFPE tissues.
(3) The “older” FFPE blocks tend to give poorer RNA concentration as compared to the tissues that were fixed only recently.
Ways to overcome these difficulties:
(1) Use RNase Zap to help reduce the amount of RNase that could be present in the surrounding (microtome/gloves). If talking is necessary during the isolation process, one could wear facemask so as to prevent the RNase in the saliva from degrading the samples.
(2) Since it’s more difficult to isolate RNA from FFPE tissues, more sections could be collected in several tubes and pooled together so as to maximize the concentration of RNA to be collected after 2 days of extraction.
(3) If given a choice, choose tissue blocks that were fixed and embedded recently (within 5 yrs) because studies have found that RNA in the blocks that were more than 10 year-old degrades significantly.
(1) During sectioning, the tissue blocks are constantly exposed to the existence of RNase in the surrounding. Hence, the RNA in these blocks might risk getting destroyed during the sectioning process and affecting the total concentration of the RNA after the RNA extraction is done by the end of Day 2.
(2) The process of fixing the tissue sample and embedding it in paraffin has caused severe degradation of the RNA. Therefore, it’s more difficult to isolate good quality RNA from FFPE tissues.
(3) The “older” FFPE blocks tend to give poorer RNA concentration as compared to the tissues that were fixed only recently.
Ways to overcome these difficulties:
(1) Use RNase Zap to help reduce the amount of RNase that could be present in the surrounding (microtome/gloves). If talking is necessary during the isolation process, one could wear facemask so as to prevent the RNase in the saliva from degrading the samples.
(2) Since it’s more difficult to isolate RNA from FFPE tissues, more sections could be collected in several tubes and pooled together so as to maximize the concentration of RNA to be collected after 2 days of extraction.
(3) If given a choice, choose tissue blocks that were fixed and embedded recently (within 5 yrs) because studies have found that RNA in the blocks that were more than 10 year-old degrades significantly.
Kangting
0503331A
TG01
Saturday, September 29, 2007
Week 14- Confocal Microscopy!
Hey..this week’s my turn, and I have interesting PICS to share from my research work. 
Q: Why do the polymers 'stick' onto the membrane?
A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!
2.5 hr after Transfection: in the cytoplasm
Q: How does my polymer enters the cytoplasm?
A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.
4.5 hr after transfection: in the nucleus!!
Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?
A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.
Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :(
But still, research is FUN! :))))
Nisha
TGo2
0503254E
In order to view my cells, Confocal laser scanning microscopy(CLSM) is used. The CLSM used is LSM 5 LIVE, the latest edition for imaging of living cells from ZEISS technology. CLSM uses laser to excite the fluorochromes, unlike fluorescence microscope(FM) which uses fluorescence light.
If you wanna know the principle of CLSM and FM, just give me a comment, as it is more like ‘physics’, afraid some of you might not understand.
One important precaution: sample preparations must be done in the dark, once exposed to light, the fluoresce intensity can greatly decrease, especially for FITC.
GREEN(FITC): polymer ( note: DNA is still bound to the polymer, but cannot be seen as it is not labeled)
The following pictures are a bit blurry because I magnify them 8-12 times from the original one.
1 hr after Transfection: on the cell membrane
Q: Why do the polymers 'stick' onto the membrane?
A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!
2.5 hr after Transfection: in the cytoplasm
Q: How does my polymer enters the cytoplasm?
A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.
4.5 hr after transfection: in the nucleus!!
Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?
A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.
Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :(
But still, research is FUN! :))))
Nisha
TGo2
0503254E
Friday, September 21, 2007
Week 13 Sharing of experiences in histopathological laboratory
In tissue processing room
- Whole mount preparation
-Completely cross-sections of a pathological specimen
-Usually too large for the ordinary cassette and require individual handling prior to machine processing
-Example: whole mount sections of prostate
Observations of prostate trimming by pathologists
-Material: Prostate cutting tool
-Whole mount is necessary as tumour is located in prostate and can only be seen under microscope.
-For whole mount processing, the timing of fixation will be extended for 41/2
hours (total 211/2 hours) to ensure tissue is thoroughly fixed.
-The dehydration steps will also be extended to ensure proper dehydration due to the size of the tissue.
-Other tissues will not be processed together with whole mount prostate as over fixation and dehydration of smaller pieces will cause the tissues to be harder.
Microtomy
1. Microtome safety (Safe Operating Procedure)
2. Handle blades very carefully when installing or removing. Follow the manufacturer’s guidelines explicitly.
3. Tungsten-Carbide knives can put through your shoes, if dropped. Be careful where your feet are positioned when installing or removing blades.
4. Store blades in a covered container that has guides to hold the blades rigid.
5. Never leave blades on countertops. Lacerations can occur when reaching across the countertop and inadvertently contacting on unprotected table.
6. When setting up the microtome, position the sample first, and then put in the blade. Never the other way around.
7. When applying the brake, ensure that it is tight. Most accidents occur when the brake slips and the operator’s hand is drawn into the blade.
8. When leaving the microtome, even for a short time, ensure that the blade guard is in place.
9. When preparing a paraffin sample for the microtome, remember to clamp the sample down tight. The movement is allowed by a loose clamp increase your risks of cut.
10. Use forceps or brushes during retrieving and transferring slides and ribbons from the blade, thereby keeping your hands free from the moving parts of the microtome.
Common faults encountered in microtomy and remedies
Causes / Remedies
Ribbon fails to form
1. Paraffin too hard / 1. Use softer wax i.e. lower melting
2. Blade too blunt point / 2. Change to a new blade edge
3. The tilt is too great / 3. Tilt the blade less
Crooked ribbons
1. Edge of block is trimmed not parallel to blade. / 1. Re-trim the block.
2. Irregularities in blade edge / 2. Try another part of the blade
Sections compressed, wrinkled and jammed together
1. Blade too blunt / 1. Change to a new blade edge.
2. Paraffin block is warm / 2. Cool the block on cryoplate
3. Blade tilt is too slight, therefore / 3. Increase the tilt
the cutting facet rubs over the block
4. Blade edge gummed with wax / 4. Wipe blade with xylene
Sections crumble and specimens fall out, mushy and soft
1. Incomplete dehydration / 1. Re-hydrate
2. Improper embedding / 2. Re-embed
Tissues turn hard and cut like stone
1. After clearing, the specimen was / 1. Nothing can be done except repeat
accidentally left to dry in air with fresh specimen
2. Paraffin bath is too hot and burns the tissue / 2. Decrease the temperature of parrafin
bath
Split ribbon or lengthwise scratches in ribbon
1. Nicks in the blade / 1. Use a different part of the blade.
2. The tilt of the knife is too great / 2. Decrease the tilt
3. Blade edge dirty / 3. Wipe blade edge with xylene
4. Hard particles (calcified materials) in the block / 4. Do surface decalcification
5. Crystal from fixative e.g. Mercuric chloride / 5. Treat tissues to remove crystals
Undulations in the surface of the section
1. The blade is not tightened / 1. Tighten all screws
2. The title of the blade is too great / 2. Decrease tilt to prevent vibration
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4. Blade edge is dull or rounded / 4. Change to new blade edge
Sections of varying thickness and skipping of sections
1. Blade not tilted enough to clear facet, / 1. Adjust the tilt
or too much and tissue is compressed until
the inevitable expansion gives a thick section
2. The blade and specimen holder not tightened / 2. Tighten all screws
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4.Cutting stroke is not regular or interrupted / 4. Maintain regular cutting stroke
Sections full of fine lines running across
1. Blade edge consist of fine serration / 1. Change to a new blade edge
2. Cutting edge is dirty / 2. Clean blade edge
Sections full of folds
1. Water bath is too hot or too cold / 1. Adjust temperature to 50 degree
Celsius
2.Blade tilt is too slight so cutting facet / 2. Increase the hilt
rubs over the block.
3. Paraffin block is warm / 3. Cool block on cryoplate
Alright i shall stop here then. :)
Ang Xiao Si Sharon
TG02
0503219H
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