First, you need a solution that would would destroy all RNases like RNase Away.

-You should clean your cryotome (all the pieces that would touch your frozen samples like, blade and adapter plate) with this solution.

-Place your sample to the cryotome and prepare it as if you would cut tissue sections for IHC.

-Shave the OCT around the tissue as much as possible.

-Start cutting, but don't use the glass piece, you don't need nice sections here.

While cutting, let the sections roll and place these rolls is a RNase free tube with a tweezer(cleaned with RNase away).

-Normally 10 sections with a thickness of 10um should be enough.

(For fibrous tissues you can use 25 sections with 4um.)

-Add the lysis solution for the RNA extraction to the tubes (Trizol works fine for me), if necessary homogenize the tissue and proceed with isolation protocol.

But you haven't mentioned the conditions like at what temperature you stored your tissues. we use to keep our tissues(100mg) in TRIZOL at -80C. It will be good for 2-3 months. But In your case may be RNA has degraded, better you take new one for RNA extraction. I am having protocol for RNA isolation.

you didn't mention about the storing process' samples are with RNA latter or in TRIZOL, yes in TRIZOL you can store for 5 months or more in -80C.

First, you need a solution that would would destroy all RNases like RNase Away.

-You should clean your cryotome (all the pieces that would touch your frozen samples like, blade and adapter plate) with this solution.

-Place your sample to the cryotome and prepare it as if you would cut tissue sections for IHC.

-Shave the OCT around the tissue as much as possible.

-Start cutting, but don't use the glass piece, you don't need nice sections here.

While cutting, let the sections roll and place these rolls is a RNase free tube with a tweezer(cleaned with RNase away).

-Normally 10 sections with a thickness of 10um should be enough.

(For fibrous tissues you can use 25 sections with 4um.)

-Add the lysis solution for the RNA extraction to the tubes (Trizol works fine for me), if necessary homogenize the tissue and proceed with isolation protocol.

Thank you so much Dr. Ayata. We did RNA extraction from tissue in OCT and also only frozen and both looks very nice. Today we are trying to do 18S as a house keeping and we will see if will get similar results. Thank you so much again

Hi Dr. Ayata and Rita,

Even I facing problem with simultaneous DNA /RNA extraction (All prep QIAGEN kit) from oral leukoplakia tissue.

I have frozen tissue, I tried crushing 20-30 mg tissue like i did for tumors, but unable to get DNA. Even trying with cryosections but unable to decide how many sections should i take.

I am unable to figure out whether the sample i am using is not enough or some thing else. Can you please suggest me, I am planing to use this samples for microarray.

Dear Priyanka,

I will start by pointing out a few technical issues. The solutions that you use, are they prepared as recommended by the supplier (ß-me, ethanol etc. additions)?

As far as I know 30mg is the upper limit of the column isolation methods. Depending on the tissue type, using less starting material could be a solution for you. Too much fibrous or fatty tissue may inhibit the columns ability to hold nucleic acids. Are you using shredders or other homogenization methods?

If you have a 1cm2 tissue surface area, 10 sections with a thickness of 10um should be enough. For smaller or bigger tissue sections you should calculate accordingly.

Dear Dr. Ayata,

I have prepared all the solutions as recommended, For homogenization i use 20G needle and syringe.

As its epithelium it has lots of keratinization and matrix, usually for tumor i take 15 micron 40 sections. But with leukoplakia i am very much confused whether i am using less or more sample.

I am using simultaneous DNA and RNA kit, so QC by nanodrop I get RNA about 200-300ng/ul but DNA is around 13-30ng/ul.

Once i will try using 10um, 10 sections

Dear Bhosale, I dont have experience with quiagen kit (I tried only once) and neither with leukoplakia tissue. But If I am you, I would try the way dr. Ayata is proposing.

Dear Dr Ayata and Rita,

First of all thanks for all the useful information on this topic, really helpful! Secondly could either of you point me in the direction of any publications you have referenced this method in, and have either of you tried a side by side test of trizol and QIA type kits to see if one is more efficient than the other ?

Cheers

Keely, We have performed a small set of side by side extractions Trizol vs column type kits and and found that Trizol consistently gives higher RNA yields. More recently we have started using Ribozol which is slightly better. Additionally we homogenize using a bead beater.