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.

(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

(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.

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.

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.

Kangting

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