Hi Pangiotis,

I've done a lot of this type of work both with cancer cells and cancerous tissue without any issues.I always used trizol with the Qiagen RNeasy kit. Please consider the following checks.

1) Prealiquot materials you'll need to use in order to minimise processing time,

2) Prechill reagents that need to be cold

3) Prelabel all tubes you expect to use.

4) Use filter tips

5) At every opportunity keep your samples on ice.

6)Take time mixing the trizol and sample, vortex robustly to ensure thourough pernetration

7) Be sure to use a DNA digest step

8) The optimal number of samples for extraction is about 6-12,unless your very efficent, more that this causes delays and over exposure at room temperature

9) Aliquot a sample of the final extraction for quantification with a Nanodrop. This will tell you a lot about possible contaminants

10) Minimise freeze-thaw of samples

11) Where possible use an RNA dedicated laminar flow hood

James

Hi,

first of all, make shure that all your materials and solutions are RNase free. Wear gloves, wash your hands/gloves and the bench frequently with 70% EtOH or RNase inhibiting/destroing wipes like RNase-ZAP. Also your pipettes before working. Work whenever possible on ice maybe in a cold room if available.

hope that helps, best,

Guido

I work in sterile conditions, with coolboxes... I wonder, if this happens due to the cells, for example, in cancer cells I have no problem, but in stem cells I have. I have tried also β-ME for protection.

Someone told me that the Trypsin could be inhibitor factor. Does anyone know anything about?

Have you tried your RT-PCR primers with a positive control.. and have you tried other genes besides GAPDH or Actin ? Its difficult to get any amplification because sometimes you even get a PCR product with degraded RNA as the products are just short fragments.. You can also test the quality of your RNA by Bioanalyzer if its possible for you..

If you get clear 18S and 28S bands and are worried there is an inhibitory factor present you could always try to amplify a sequence from one of those bands as a test for whether your RT and PCR are functional in those samples.

Dear Shrey, I have tried to amplify high length of 189S-28S (1500bp), but there was no product. Dear Adam, the bands are not always clear, when I run denaturate-agarose gel. But even in samples that I got clear bands for 189S-28S, the othe genes were not amplified.

I think that maybe the cells are not break well. I've tried many detergents (TRITON, Sonication, Proteinase K), but always the same..

If your RNA samples are non-degraded (check it by using a Bioanalyzer if possible) I think that the origin of the problem is with your RT-PCR (check your primers for example). Best, Geraldo Passos

I suggest to use another method which I use with my bacteria (Frankia), this methods is efficient and give high yield from mRNA and very low yield from 16s rRNA.please see the attach paper.

You can start by running the total RNA on a gel to see whether it is actually degraded. If the RNA is good you should see two bands corresponding to the 28S and 18S rRNA. If you do see these bands, then your problem may be with inhibitors or with your RT-PCR conditions. With partially degraded RNA, you will see these bands as well as smears from these toward the bottom of the gel. If you only see a smear then the RNA is very degraded.

I have done a lot of RNA extractions without any problems. I clean my pipettes with RNase-Away (Invitrogen) prior to use, and use tips and tubes that are RNase free. (Many tips and tubes, such as those from Axygen, come RNase-free and don't need any other treatment. If you rack your own tips, do it carefully with clean gloves that have been rubbed with RNase-away. Do not autoclave your RNase-free tips and tubes.) Use clean gloves. Once you touch your lab bench, or the centrifuge, or anything that is not RNase-free, your gloves are not clean anymore. Make sure you don't put anything that is not RNase-free in or over your tubes once the protein-denaturing agent (Trizol, etc.) is removed from your sample. I usually hold the tubes at a 45 degree angle when adding or removing material so that nothing can fall into the tubes from my hand or pipette.

Dear Panagiotis

Which Trizol do you use ? (Normal or HC). How many cell you harvest for extraction? can you lower the amount of clear layer taken after centrifugation? Trizol or other similar reagent are appropriate for large amount of cells and because of lesser manipulation of almost RNA quality are perfect. All the suggestions that other friends were recommended are exactly right. Despite the other recommendation keep in mind the possibility of extracted RNAs with guanidium isothiocyanate. It is a well known inhibitor of PCR reaction. I hope every thing is going well.

Hi Pangiotis,

I've done a lot of this type of work both with cancer cells and cancerous tissue without any issues.I always used trizol with the Qiagen RNeasy kit. Please consider the following checks.

1) Prealiquot materials you'll need to use in order to minimise processing time,

2) Prechill reagents that need to be cold

3) Prelabel all tubes you expect to use.

4) Use filter tips

5) At every opportunity keep your samples on ice.

6)Take time mixing the trizol and sample, vortex robustly to ensure thourough pernetration

7) Be sure to use a DNA digest step

8) The optimal number of samples for extraction is about 6-12,unless your very efficent, more that this causes delays and over exposure at room temperature

9) Aliquot a sample of the final extraction for quantification with a Nanodrop. This will tell you a lot about possible contaminants

10) Minimise freeze-thaw of samples

11) Where possible use an RNA dedicated laminar flow hood

James

Does β-mercaptoethanol help in avoid RNases?