WEEK 5 ATTACHMENT SHARING

Heya guys……zahirah here….i’m currently attached to clinical pharmacology lab with michelle…if u guys think that we are in clinical lab..then u guys are super duper wrong..hahaha…well we are actually in a research lab doing studies on the genes of different ethnic group and see how it might affect the pharmacodynamics and kinetics of the treatment…think BPHARM….but then our lab deals with the major techniques in MBIO which is PCR and DNA sequencing…since we are together and to avoid u guys from reading the same thing twice….we will try our best to post up on different things….:)

Title: Running Gel electrohphoresis
Purpose: To determine the optimal temperature of the primers that we are going to use for the research by looking at the number and intensity of the bands.

REFER TO MICHELLE’S BLOG (http://jammys-bms.blogspot.com/) FIRST FOR THE OPTIMIZATION OF PRIMER

Before we run the gel…of course we need 2 make the gel first and it is prepared while waiting for the PCR to finish..save time a bit….soo..here’s the ‘ingredients’ and ‘recipe’ for the gel

For making agarose gel
Materials:
Agarose powder 1X TAE buffer Ethidium bromide Combs Tray
Conical flask Measuring cylinder

Method:
1.Prepare the tray by covering the open ends and pour DI water to measure how
much volume of TAE buffer is needed
-the volume needed is dependant on how thick you want your agarose to be
2.Pour away the DI water and place the tray in the fume hood
3.Calculate the amount of agarose powder needed
-this is calculated using the formula:
% of agarose gel x vol. of TAE buffer = weight of agarose powder
-since in this lab we use 1.5% agarose and the volume of TAE buffer needed is
70ml
thus: 1.5% x 70ml = 1.05g
4.Weigh 1.05g of agarose powder and put it in the flask
5.Measure 70ml of 1X TAE buffer
6.Pour the 1X TAE buffer into the flask and swirl gently
7.Place it in the microwave for 50 sec
8.Once you get a clear solution, bring the flask to the fume hood and swirl for a
few seconds first
9.Add 3.5ul of ethidium bromide into the flask after the solution has cool down a
bit
10.Calculation for vol. of ethidium bromide needed:
Stock conc: 10 000ug/ml
Dilution conc needed: 0.5ug/ml
M1V1=M2V2
0.5ug/ml x 70ml = 10 000ug/ml x V2
V=0.0035ml = 3.5ul
11.Swirl the flask gently to obtained homogeneity
12.Pour the solution into the tray and place the combs

Note: the volume of 1x TAE buffer, agarose powder and ethidium bromide and the number of combs needed is dependant on the size of the tray/agar. For this one, it is for a normal size agar that is able to hold two combs. The thickness of the comb is also dependant on what product we are going to run. For primers, we use a comb with big and thick wells as we are going to load all the PCR products.
For running gel electrophoresis
Materials:
6x loading dye PCR products 100bp ladder 1.5% agarose gel

Method:
1.Place the agarose gel into the gel electrophoresis machine
-ensure there’s enough TAE buffer to cover the gel
-the wells is placed at the negative part of the machine
2.Pipette 2ul of 6x loading dye into each PCR tubes and mix
3.Load the samples to each corresponding well(starting from the 2nd well)
Eg. 1st PCR tube-to the 2nd well
12th PCR tube- to the 13th well
-must ensure that we don’t jumble up the tubes and the loading part because
each tubes represent the product that has undergone different annealing
temperature
4.Pipette 2ul of 100bp ladder to the 1st and last well( after the 13th well)
5.Run the gel at 120V for 15-20 min

++Initially we want to post up the pictures we obtained after gel electrophoresis but when we ask our supervisor for permission, he did not give us a clear cut yes….soo sories guys…but u can refer to your MBIO lecture notes the topic after cloning…the bands we obtained are almost similar to that…

A BIT ON LMQA...
All staff is required to attend safety modules which even include a module on centrifuge safety..at the end of each module, we actually have to sit for a test and even do assignment. All the work done in our lab are pretty manual..except for those that must be done by machines like DNA sequencing, etc...not that advance like nisha's where there's even an automatic pipette filler..our fridge actually have warning that says may contain biological agent because we do store blood from patient's in the same fridge we store our reagents...and there are some labs that have the radioactive symbol(the three triangles) because that particular lab deals with radiation/radioactive materials or they used to deal with it.....

so my posting is till here......shall continue the nxt time i post....wishing everybody all the best for your SIP/MP and enjoy....tata...:)

Week 4 Attachment Sharing

Greeting to all fellow BMT course mates!! It is time for me to share my experience in SIP with you all. Let begin!!

Basically, I am attached to a research lab, which revolves around gene expression and its relation to certain disease. Concurrently, I am working on a particular gene, which might be related to diabetes. In this research, most of time I am dealing with a lot of MBIO and MCT stuffs, for instance things like PCR, real-time PCR, cell culturing and DNA sequencing.

Introduction to real-time PCR
Real-time PCR also refers to kinetic polymerase chain reaction. It is a unqiue technique commonly used to amplify and at the same time qualify the amount of a specific DNA of choice in the sample. In fact, it detects the amount of target DNA based on the amount of fluorescence release from its probes during the PCR process.

Principles of real-time PCR
Let recall our biochemistry and molecular biology knowledge! As we have learnt in lessons, PCR amplified double- stranded DNA by adding Taq DNA ploymerase, dNTPs, DNA templates, PCR buffer, last but not least the primers (both forward and reverse). Hence, when these solutions come together in PCR, the primers will anneal to the flanking sequence of the denatured DNA. Followed by extension by Taq DNA polymerase with the aid of dNTPs.

However, in the case of real-time PCR, probes are also added into the PCR master mix solution. These probes comprised of a reporter and quencher dye each at both ends. In addition, these probes sequences are complementary to the target DNA sequences. Therefore if the target DNA is present in the sample these probes will bind to it specifically and form a hybrid. However, the 5’ to 3’ nucleolytic activity of the Taq DNA polymerase will cause the hybrid (probe- target DNA) to cleave in between the reporter and quencher dye. A new extended strand of DNA will then displace the fragmented probes as the ploymerisation continues. Since the 3’ end of the probes is blocked, there will not be any amplifying of probe. The results is then read and recorded by software. The higher the fluorescence intensity indicated a higher amount of target DNA and vice-versa.




Designing the probes
Unlike other labs, my lab synthesizes our own probes. In order to synthesizes a probe there are certain guidelines to follow, such as
· GC contents of the probe must be within 20-80% range
· Identical nucleotide must be avoid
· Avoid a G nucleotide at the 5’ end
· Choose probes that have Cs instead of Gs
· The melting temperature should maintain at 68- 70 °C
· Probe sequence must be near to primer but not overlapping it


TaqMan Allelic Discrimination Kit
TaqMan Allelic Discrimination Kit is one of the most important kits I used during my research. It employed the principles used in the real-time PCR; only different is that it consists of two types of different probes instead of one. The two type of fluorescence probes used is TET (6-carboxy, 4,7,2,7’- tetrachlorofluorecein) while the other is FAM (6-carboxy fluorecein). These two probes is covalently linked to the 5’ end of the probes. Both type of probes will bind to their respective target DNA template (in this situation is two alleles) and form a hybrid. Fluorescence signals given by the reporter will only be detected when the DNA template is polymerized.

Take example; TET represents allele 1 and FAM represent allele 2. If a sample DNA template undergoes real-time PCR and high TET fluorescence signal is detected. This could mean that the individual is a homozygous for allele 1 and vice – versa for allele. However, what if both the signals are equally high?? Hmm…


Ans: That would indicate that the individual is a heterozygous (having both allele 1 and 2) (have you got it right?? Hehe)

Procedure for real-time PCR
1. Prepared the Real-time PCR master mix by adding the following:

· dNTPs,
· PCR buffer
· Probe
· RNase free water

2. Vortex the solution briefly
3. Add in the Taq DNA ploymerase
4. Inverted the centrifuge tube 2 to 3 times to mix the solution
5. Aliquot 9ml(for my lab) into each well in the 96 wells reaction plate/ MicroAmp optical tube
6. Close the plate tightly with the MicroAmp optical caps
7. Briefly centrifuge the plate (this is to allow the droplets cling on the side to settle)
8. Place the plate onto the thermal cycler block
9. Ensure that the real-time PCR cycling is in this manner:

· 25 ºC for 10 mins
· 48 º C for 30 mins
· 95 º C for 5 mins
10. Remove the plate and stored in fridge

Tips for real-time PCR
Here are some tips to reduce the chances of getting a false positive result. High DNA concentration from sample, amplicon carryover from previous PCR or positive control can lead to false positive results. In such case, AmpErase UNG (uracil N-glycoslase) treatment can be applied. This is because when UNG (uracil N-glycoslase) solution is added, the dTTPs are substitute by dUTPs in the PCR process. Hence it prevents the reamplification of the carryover in subsequent process by degrading the misprimer or non-specific PCR products via enzymatic reaction.

Another useful tip for PCR process is the addition of Q solution. Q solution changes the melting temperature of the DNA template during the PCR process. Therefore increase the specificity of the primers anneal to the DNA template during the PCR process. Besides that, it also increases the chances of achieving successful amplification and more PCR products.

By the way, if you have any question feel free to give me your comments.
Till then enjoy your SIP and see you all in the upcoming week!!

Avery, May lee(0503292E)
TG02

WEEK 3 Attachment Sharing

Hello guys, it's my turn to share my SIP experience with you! =)

Anyway, for your information, i am currently working at a Oncology (cancer) research institute and everyone in my group is actually pretty much working on their own projects though we have general lab meetings every alternate weeks to update each other on our research progress etc etc.

As for me, i am focusing on a particular gene (let's just call it gene A) to see if there is an over-expression of it in prostate cancer. If there is a huge amount of gene A found in the prostate cancer cells, we can then make assumptions that perhaps this gene A has something to do with encouraging tumour angiogenesis or cancer cell proliferation etc etc. From it, we will then be able to find out if we could come up with an inhibitor that can block the action of the proteins (translated from gene A) and hopefully treat this cancer! (ok, this is the simplified version of my job scope. it's so much more complicated if i really want to elaborate but i will just leave it as that ok? =] )

For the past 3 weeks, i have been trained on Immunohistochemistry (IHC) which is very commonly used in cancer research to allow one to check if a particular protein is present/over-expressed in the cancer cells of that particular organ of the body. If you can still recall what we learnt during HistTech, it's 90% exactly the same as the staining we did =)

IHC: Materials and Methodology

1) As tissue samples from hospitals normally are formalin-fixed/paraffin-embedded sections, deparaffinization was done in 3 consecuetive xylene baths for 5 mins each

2) The slide was then dipped into 2 changes of absolute alcohol for 5 mins each as well.

Significance: To remove xylene from the slides and to prepare the sections for rehydration. This is because xylene and water are NOT miscible.

3) Rehydration was then performed as the slide was dipped in 95% alcohol (2 changes, 5mins each) before it was placed in 75% alcohol (2 changes, 5mins each). Rehydration is considered complete when the slide was rinsed with tapwater.

4) Pre-treatment was carried out as the slide was immersed in Ag Retrieval Buffer before it was placed in the Mega Microwave Histoprocessor at 117 degree Celsius for 30 mins.

Significance: When you fix a tissue sample using formalin, what it does is to denature proteins by coagulation, by forming additive compounds or by a combination of the 2. As a result, confomational changes in the protein structures will then occur to inactivate the enzymes and preserve the cells/tissues. Therefore, this pre-treatment process is to "revive" the immunoreactivity of the Ag to an immunogen. This process is aka "epitope retrieval" process.

5) The slide was cooled for 20mins before it was dripped dry and encircled using a Dako Pen

Significance: Dako pen is able to "trap" whatever reagents used on the sample within the encircled area so we don't have to use too much reagents and waste them.

6) Peroxidase block was added for 10 mins to the section to block any endogenous peroxidase that was produced by the cells.

Significance: Our secondary Ab will be labelled with Horseradish Peroxidase. Hence, we don't want any false positive results because of the presence of endogenous peroxidase.

7) Slide was washed to remove any excess peroxidase block reagent.

8) Ready-to-use Protein Block was used on the slide for 1 hour

Significance: Remember we have learnt about the "blocking" step in MBio last semester? It works in the same theory: to prevent non-specific binding.

DO NOT WASH SLIDE THIS TIME ROUND because even if you feel that the protein block might block the antigens in which the primary Ab have to bind to, this is not true. According to my senior, as the primary Ab used is more specific to the Ag than the protein block, it will have a higher affinity and the former can eventually replace the protein block. Hence, the end results will not be affected.

9) Primary Ab was then added to the slide for 1 hour. For every different kind of Ab used, we have a specific dilution for it. In case you are curious to know, i am using 1:5000 dilution =)

10) Washing was done this time to remove excess Ab

11) Secondary Ab labelled with HRP was added. Since my primary Ab is raised in mouse, my secondary Ab is an anti-mouse solution yup!

12) Washing carried out again

13) Chromogen and DAB was then added for just 5 minutes.

Significance: DAB will oxidise when it comes into contact with the air in the environment causing polymerisation and increasing its staining intensity. Chromogen is the substrate that will bind to the HRP to give us the signal we want.

14) After the slide was rinsed with tap water, it was counterstained using only Mayer's Haematoxylin.

Significance: As recent research has shown that protein A (from gene A) will be localised only in the nucleus, we only have to use haematoxylin and can save on our Eosin dye.

15) Dehydration was then carried out as the slide was dipped into 75% alcohol (2 changes, 5 mins each), 95% alcohol (2 changes, 5 mins each) and lastly absolute alcohol (2 changes, 5 mins each)

16) The slide was placed in the fume hood for 10 mins for drying to completely remove any water present on the slide/sample

Significance: water and xylene = NOT miscible

17) Lastly, the slide was placed in xylene for 3 changes for 5 mins each before it's mounted using DPX

Example:

Figure 1. Prostate cancer tumour sample expressing gene Z (Nucleus gives dark brown coloration). Note that only Mayer's Haematoxylin was used because researchers already know that gene Z staining is mainly localised in the nucleus.

Image is taken with permission from fellow co-worker.

Hope i didnt get my concepts wrong. LOL. correct me if you think i am inaccurate in any way yup =) Anyway, hope you guys are enjoying SIP over at your side.

Psst: should u have too much time to spare, u can join me for lunch coz i m alone like nisha as well! haha.

KangTing TG02

0503331A

Week 2 Attachment Sharing

Hey there, i miss everyone because I'm alone here..haha, here's my part, if you don't understand, just ask me, if I never reply means I don't know what's the answer. lol

About my attachment

I’m attached to a research institute, in which all levels have research labs and different focus areas. I will be working on a paticular level throughout my attachment, and this level focuses on delivery of drugs, genes and proteins. Each student in my lab who are mainly university students and they are attached to a mentor, including myself and we are assigned onto a specific delivery. The major problem in current researches done throughout the world on such deliveries is that, the delivery efficiencies are low. Thus, we are trying to enhance the delivery of drugs and genes into cells for future cancer therapy and treatment of infectious diseases. Majority of the publications done by IBN are on cancer therapy.

Reseach Focus

My delivery focus is genes. These genes can be plasmid-encoding ones or siRNA. I’m working on how efficient the tranfected cells express the genes delivered to them and also the percentage of viability. Finally a comparison is done using different cell lines and nanocarriers. Let me explain the principle first.

Principle of the Project

This lab synthesizes nano-sized cationic polymers as carriers for the genes. The polymers have nitrogen group( NH₃⁺) which are positively charged that bind to the negatively charged phosphate group(PO₄^-) in the DNA. They form a transfection complex which will then be used to tranfect the cells. The uptake occurs via endocytosis. I will prepare the polymers and DNA solutions based on very confusing calculations in which I took almost a week to understand. The tranfection complexes are prepared in different conditions and pH(5&7). Conditions basically mean different nitrogen to phosphate ratio(N/P ratio: 0-40). I usually do 9-10 conditions. These are mainly done to determine the optimal concentration of the transfection complex and at what conditions do cytotoxicity occurs.

Lab techniques

Since I’m dealing with cells, I need to master all the cell culturing techniques and maintain my own cells. There are quite a no. of machine that I must learn to operate. Apart from that, I will have to prepare all my reagents by myself (labeled with my name) throughout my experiments and strictly no sharing.

Culturing and seeding of cells

After preparing all reagents and subculturing, I will seed the cells on 24 well plates or 96-well plates depending on which cell lines. Some cell line that I handle are HepG2, HEK 293, heLa and MCF-7 & etc. On the next day, I will do gene transfection on 1 cell line. The cell must meet the desired density, thus cell counting must be done using haemocytometer. Each condition has 4-6 replicates, thus every row of plate is for 1 condition. Then I will repeat the whole thing for another pH. The cells are then incubated in the CO₂ incubator for 4 hrs and then the medium are changed to stop transfection. I will then need to wait for 3 days to do analysis using MTT assay and luciferase analysis assay.

Zeta potential and particle size

During the 3-day incubation, I will measure the zeta potential and particle size of the transfection complexes. Zeta potential basically means the surface charge of the particle. Since the cell surface are slightly –vely charged due to proteogylcans in the cell membrane, the complex must be more positive to enter the cell. However it must not be too high, because if too much complex enter the cells, cytotoxicity can occur. Apart from that, the particle size is also measured. It cannot be too large as they need to enter through the nuclear pores which are only 10nm in diameter. Both can be measured using a nanozeta and size analyzer based on different conditions and pH.

Assays for transfection efficiency and cytotoxicity & Total protein measurement

After 3 days, I can then do my analysis. I have not learnt the MTT assay procedures which is done to measure the cell viability, so I will explain about the luciferase analysis assay which can consume the whole day(seriously). This is done to determine the transfection efficiency(how many cells take up the complex). The DNA that I used for the complex is a luciferase-encoding plasmid. Cells that have been transfected will have the luciferase gene and can express the luciferase protein. The cells are then washed and lysed using lysis solution and further scrapped using pipette tips. This must be done properly as it will greatly affect the results. I took 3.5 hrs to do 4 24-well plates.

Then, I will measure the light intensity by adding substrate to the luciferase protein in the sample using the luciferase analyzer machine. A BSA standard is also done to convert the light intensity value to protein concentration. Apart from that, total protein content is also done to determine what is the percentage of luciferase protein over the total protein expressed by the cells. Both the BSA standard and total protein content are measured using 96-well plate reader.

From the beginning to the end of the experiment, I do have lots of excel sheets and graphs to plot. I should say that the steps and procedures are quite tedious and time consuming. Yet, IBN is good place for students like us because they give you the freedom to conduct any experiments you want, so long as it is within the focus area of the lab.

As you can see I did not explain about the step-by-step procedures for each phase because it is very long, so if you want to know, just send me a comment. I will blog more about result analysis and siRNA on my next post.

A brief one on LMQA

A*STAR divisions especially IBN put great emphasis on safety in the labs. Almost everyday, I do receive emails on safety aspects and every month, I have to attend safety briefing. I have an RFID card that I must always carry with me, so that if there is any emergency occurence, they can track where I am electronically. Apart from that, each lab has a SOP and incident occurrence box at the entrance. This white box contains the necessary documents to operate the machines, and also filing of accidents that have happened in the labs. Every 2 weeks, a safety officer will regularly check the boxes if all documents are present. Plus, at every level there is a MSDS room where you can find all the A-Z details of the chemicals. We are also given a small card containing a list of safety contact officers in case if there is any accident occurrence.

Regards,
Nisha Bte Mohd Rafiq
0503254E
TG02

Student Internship Programme Sharing

Hi everyone, how's your first week of sip? Do share with me, ok?

I was being attached Histopathology/Cytology with June, Wing Fat and Desmond. For whole of 20weeks, we are being sent to the different stations of the histopathology laboratoy. For the first month, i am stationed at special staining, after which is cytology then processing next it will be embedding and microtomy and lastly we will take shifts to help in all the stations of the lab.

For special staining, i have learnt how to use and care for H&E (Haematoxylin&Eosin) machine and Ventana special stainer machine. It also includes batch collection and mangement of slides. Im also expected to pick up special histological staining techniques to produce good quality stained sections.

I also read up quality management system, environmental management(ISO 1400I requirements), emergency actions, safe disposal of waste, sources of contamination and suggestions to prevent it from the medical technologists in the lab. Some of these can be found in our LMQA notes. We do have to follow good lab management in order to have quality assurance.

Histopathology Lab Process
1) Customer services (hospitals)
2) Histology lab reception (specimen identification and sorting by medical technologists)
3) Accessioning (numbering and sorting request forms)
4) Tissue Processing
5) Microtomy
6) Special staining/routine H&E stains (Haematoxylin & Eosin)
7) Mounting
8) Sorting and labeling of stained stains
9) Dispatch slides
10) Reporting by pathologist
11) Typing
12) Verification
13) Electronic signed-out
14) Reports dispatched-out

Hot plate
- Up to 30 tissue samples slides can be placed on the hot plate
- Sample slides are placed on the hot plate for at least 3minutes
- Small tissue sample slides must be totally dried before placing onto hot plate so to prevent floating

Routine Auto-Stainer
For routine Haematoxylin & Eosin steps involves:
1. Xylene
2. Xylene
3. Absolute alcohol
4. 95% alcohol
5. 70% alcohol
6. Water
7. Haematoxylin
8. Haematoxylin
9. Water

Ventana automated special stainer
- An automated system that is barcode-driven with optimized protocols
- Helps to increase productivity and efficiency
- Image link: http://www.ventanamed.com/ (NEXES special stain)
- Special stain wash solution is required to spray onto the slides to hydrate the tissue samples to prevent them from drying up as it may affect staining
- Example of special stains carried out: PAS(Periodic Acid Schiff), PASD, GMS(Gomori’s Methenanine Silver Nitrate) for fungus, Re (Retic), Fe (iron), Giemsa, Steiner, Alcian Blue
- Slide tray holds up to 20 test slides
- Reagent tray holds up to 25 reagent
1) Key in barcode labels and protocol(test selected) for the slides that are being used
2) Put slides in slide tray
3) Load reagents required
4) Put back reagent tray and open up all reagents’ cover
5) After which, off preheat as preheat is on before machine is used
6) Then click Run
7) Make sure all criteria of pre-run checklist is met
8) Key in total number of slides being processed
9) Run time is shown
10)When the staining process is done, remove reagents and slides
11)Click sign off
12)Slides ready

This is one of the special stains that is being done manually: Test for tuberculosis (TB)- Ziehl Neelsen
TB test/ Zn : stains for tubercle bacilli head : red
Steps
1) Sections to water
2) Commercial TB colour (5mins)
3) Wash in water (running water)
4) Differentiate with 1% acid alcohol (until colorless/light pink)
5) Wash in water
6) Counterstain with 1% Loeffler’s methylene blue (5-10sec) until sky blue
7) Wash in water and go straight to 95% alcohol to control blue color (if very blue, use 70% alcohol)
8) Dehydrate, clear and mount

Sorting of slides
- 10% of the 30 slides are checked for completeness and if they are satisfactory; if they are unsatisfactory or did not meet the guidelines, they will be sent for re-cut (corrective action)
- Is part of quality check
- This is to safeguard the patients’ interest
- Check ready slides against tissue blocks to ensure that both are from the same source
- Request forms that are sent out with ready slides are to be stamped by Amano Pix 3000 with date and time

This is what i have went through in my first week of SIP and this shall be the end of my first post. I shall update more the next time.

Ang Xiao Si Sharon
0503219H
TG02