RNA Isolation Paraffin Block

[proatpcr]

How can I isolate good quality RNA for microarray analysis from paraffin blocks?

cheers

[danfive]

You may want to consider any of the following. The DNA extraction techniques should work for RNA as well, but you may want to add DNAse digestion at end.

Originally Posted by danfive

from [Only registered users see links. ]

Section 1. Non-heating DNA Extraction Protocol

Cut paraffin block at 10 µm and collected in an autoclaved plastic microtube (1.5 ml).
Add 1 ml xylene to the microtube containing tissue sections for 30 min for two changes,
Add 100% and 75% ethanol for 30 min with two changes
Wash with PBS for 15 min with two changes
Add 500 µl of lysis buffer (proteinase K 20 mg/ml, 50 µl, 1 M Tris-HCl solution 10 µl, 0.5 M EDTA 2 µl, 10% SDS 100 µl, and distilled water 838 ml)
Incubated at 52°C overnight until all tissue fragments were dissolved completely
Add 500 µl phenol:chloroform:isopropanol alcohol at 25:24:1 to the de-waxed tissue
Mix by vortex
Centrifugation at RT, 12,000 x g for 10 min
Transfer the supernatant fluid to an autoclaved microtube using a 100-µl pipette
Add one volume of chloroform to the supernatant, mixed by vortexing
Centrifuged at 12,000 x g for 5 min.
Carefully remove the upper aqueous supernatant to another fresh microtube
Adding 0.1 volume of 3 M sodium acetate to the new tube
Mix by vortexing
Add 1 volume of isopropanol, and incubate at -20C overnight.
The precipitated DNA was centrifuged at 12,000 x g at 4C.
Discard the supernatant fluid and wash once with 75% ethanol.
Collect the extracted DNA after further centrifugation.
Dissolve the final yield of DNA in 50 µl distilled water after drying completely in a hood.


Section 2. Heating Protocol for DNA Extraction

Overview:
According to Shi et al, heating before following the standard protocol above can get high DNA yield.

Materials and Reagents

Universal Buffer Solution
Add 28.6 mM of each chemical
Citrate acid,
KH2PO4,
H3BO3,
Diethylbarbituric acid.
Adjust pH to 9 using 0.2 N sodium hydroxide

Procedure:
Add 500 µl universal buffer solution at pH 9 to a microtube containing a 20-µm tissue section
Heat at 120°C using an autoclave for 20 min
Allow the tube to cool for 5 min
Follow the standard protocol above but omitting the enzyme digestion step

Reference:
Shi et al, Journal of Histochemistry and Cytochemistry, Vol. 50, 1005-1011, August 2002.

Originally Posted by danfive

Cut 10-20 um sections. Heat at 60 degrees for 30 minutes, immerse in Hemo-De or Citrisolve, for a total of 15 minutes with 3 changes. Rehydrate by immersion in graded ethanols (100%, 95%, 75%, 50%) then water for 5 minutes each. Then immerse in PBS for 5 minutes. Ready for Proteinase K.

Originally Posted by danfive

RNeasy FFPE Kit----Qiagen kit especially for FFPE.
You could also deparaffinize (heat at 60C), hydrate (graded EtOH 95%-50%), rinse in PBS, then digest with Proteinase K and either use the regular Qiagen RNeasy kit or Trizol extraction methods.