A couple notes: First, make sure to always always always heat the RNA before synthesis. I always heat at 70c for 5 minutes, then immediately cool on ice. The quick cooling is essential, as otherwise the RNA will re-anneal into double-stranded segments that won't amplify. For strip tubes, I use a metal block that I place on ice, or alternatively, an ice-water solution... otherwise, many tubes won't contact the ice in the bucket. Without this step, your yield will be terrible.

#2 -- I have found the RT reaction to be exceptionally sensitive to water quality. I have taken to buying bottles of RNAse/DNAse free water (they're not that expensive -- probably $30-40 for a liter). I aliquot them out when they come in, and one liter probably lasts a year for RT-PCR and other molecular uses. I started doing this because I had several instances when the lab water killed the reaction -- probably either the wrong cartridge on the purifier, and in the case of DEPC, there can be all sorts of additives in the steam source for the autoclaves. It's an easy and not very expensive way to cut out one potential source of failure...

#3 -- As noted above, it's worth checking to make sure the RNA integrity is okay. The simple way to do this is to run it out on an agarose gel. You don't need to get super fancy, just pour a gel in a clean tray, and make up fresh TAE buffer (the EDTA will help keep the RNA intact long enough for this purpose).. Take a ug or two, heat and quick cool as described above, then load it on the gel and run it. In a good sample, you should see two bright bands of modestly small molecular weight that correspond to the major ribosomal fractions, occasionally a third or fourth band as well. (The apparent sizes change from species to species... ) If you don't see the rRNA bands, it likely means that the RNA is in poor condition.

Hi,

I would suspect my RNA rather then other kit components. It should be RNA degradation. I experience similar problem, I started from scratch and things started working as usual.

Did you change any single solution?

No, I used the same kit to redo the first cDNA synthesis.

Question for you: Am I correct in assuming that you are running the real-time PCR? If so, do you see any amplification on your internal control gene (i.e. beta actin, 18s, etc)? If not, then I would do following: 1). Check your RNA and make sure your RNA is high quality and intact. I would suggest you look at UV absorbance curve on Nanodrop and see if they look good (single peak with good 260/280) I would also run RNA on the agarose gel and see if RNA is intact before the cDNA synthesis. 2). Run "regular" RT-PCR with some control gene after cDNA synthesis and see if your cDNA is good (save $$ on dye/probe etc). and 3) test your real-time reaction using known cDNA or some sort of standard (plasmid, etc). I would suggest you serially dilute your cDNA/standard and see if you can detect changes in amplification. My answers seem mundane and I am sure some people may have already told you, but they usually work.

Have you checked your primers before?

Yasuhiro: so

1) according to 260/280 ratio RNA is high quality, but I did not run RNA on agarose gel order to see if it is intact. My idea is that RNA is degraded.

2) I run also beta actin and that band was very weak so the only explanation is that RNA is degraded.

your reagents for RT-PCR could be contaminated. Try with new one and also consider for a new primer for your gene.

May I suggest if you can take a look @ our website http://www.isletbiology.com

Go to section Lab SOPs see Chapter 9 and Chapter 15. At the end of Chapter 15 a link is provided to an excel file with formulations to calculate RT-PCR related calculation. It takes direct input from Nanodrop data to do all calculations.

Sarang

Hi, I agree with some of the folks here that the issue might be in your RNA...Could you run a fresh RNA prep?

I tried again after I changed water and new kit for RT-PCR, but still very very weak signals for X gene and for beta actin.

Tomorrow I want to run this guys on a agarose gel.

But actually I got totally lost, I have no idea where is coming the contamination from.

I couldn't find the SOPs

Anyway from the nanodrop calculation I used 3 ug of RNA which is a lot.

Hi Daniela,

Before you do not run your samples (RNA) in a gel, you can not conclude that you have RNAses contamination. Run a few samples in a gel (new gel and clean buffer, as RNAses can digest your samples while they are running) and check for the two expected ribosomal bands. Let us know how this looks.

I woulds suggest cleaning your pipettes, bench and everything. I usually reverse transcribe (1.5 ug should be enough) my samples next day or as soon as I can, as the less time you give RNAses to work the better.

Good luck!

Daniela:

1) Use Internet Explorer only for browsing this site. It does not show up on Firefox or Chrome

2) We always used 1 ug input which works fine. Higher concentration samples were diluted close to 1 ug.

I propose you to perform an agilent assay to confirm the RNA intergrity and quality, and can carry out you PCR with a salt (MgCl2) dilution series (e.g. Concentrations : 2 - 2.5 - 3 - 3.5uM).

Good luck.

I don't think RNAse is the problem. RNA is more stable then many people think. What is important is to use the RNA directly without freezing for making cDNA. After freezing the transcription often don't work really good.

have you made a dilution of your cDNA because if you did you may have use a wrong plasticware!!!!which is sticking the nucleic acid

For storage of RNA I would recommend you to use low binding tubes f.g.: http://eshop.eppendorfna.com/product/view/822/#producttabs-1

Cheers, Nadine

Try cleaning the bench with RNase Zap solution. Use Qiagen Kit and also use RNase-free water and I agree with Sven. It is always best to do RT the same day once the RNA is isolated.

Hi Daniela, If you use One-step RT-PCR, not applicable, otherwise, in Two-step RT-PCR please check out the reverse primer. Besides, if you run negative and positive controls with your samples, some factors like enzyme and so will be ruled out. By the way, in the end, you might retrieve all RNA extraction solutions in a RNAase free medium. Good Luck. Regards.

Dear Daniela

I used DEPC water when I did mRNA , but I used for Macrophage cells. I had best result. you can try

You have to check integrity of your total RNA (you will not see it if you had purify mRNA). Nanodrop only mesured the quantity of nucleotides present in the mix but not the integrity of your RNA. Some pigmented cell lines could be harder to amplify because of inhibition of the Taq. It is a good idea to aliquot your RNA because freeze/thaw cycles are not good for RNA like RNAse. Check Ambion website to have hints of how to work with RNA.

Good luck

Check if you are using enough RNA for cDNA synthesis. Also, did you try multiple primer-sets (e.g. some housekeeping gene, besides your gene of interest) on the fishy samples, to rule out primer-related problems?

I run RNA samples on agarose and they were totally degraded. So now I know why RT-PCR didn' amplify enough.

Extract RNA afresh every time you make cDNA.....

A couple notes: First, make sure to always always always heat the RNA before synthesis. I always heat at 70c for 5 minutes, then immediately cool on ice. The quick cooling is essential, as otherwise the RNA will re-anneal into double-stranded segments that won't amplify. For strip tubes, I use a metal block that I place on ice, or alternatively, an ice-water solution... otherwise, many tubes won't contact the ice in the bucket. Without this step, your yield will be terrible.

#2 -- I have found the RT reaction to be exceptionally sensitive to water quality. I have taken to buying bottles of RNAse/DNAse free water (they're not that expensive -- probably $30-40 for a liter). I aliquot them out when they come in, and one liter probably lasts a year for RT-PCR and other molecular uses. I started doing this because I had several instances when the lab water killed the reaction -- probably either the wrong cartridge on the purifier, and in the case of DEPC, there can be all sorts of additives in the steam source for the autoclaves. It's an easy and not very expensive way to cut out one potential source of failure...

#3 -- As noted above, it's worth checking to make sure the RNA integrity is okay. The simple way to do this is to run it out on an agarose gel. You don't need to get super fancy, just pour a gel in a clean tray, and make up fresh TAE buffer (the EDTA will help keep the RNA intact long enough for this purpose).. Take a ug or two, heat and quick cool as described above, then load it on the gel and run it. In a good sample, you should see two bright bands of modestly small molecular weight that correspond to the major ribosomal fractions, occasionally a third or fourth band as well. (The apparent sizes change from species to species... ) If you don't see the rRNA bands, it likely means that the RNA is in poor condition.

As many of them suggested, better you through all reagents that could be contaminated and start from the new rna extraction, eventually it will save tome and money!

Chance of DEPC water contamination is low unless it is exposed more to RNase contaminated atmosphere

If you are not getting result with same samples then consider RNA integrity. If the samples are different from your previous samples then it might be that RNA secondary structure is inhibiting RT reaction.

As people suggested here denature RNA at 70 degrees and add some high density substance in reaction mixture.

Hey guys, I solved the issue even if it sounds so weird!

I have washed cells three times before adding Trizol and now RNA is intact and no more degraded.

Thank to all for helping me!