Spec your RNA (nanodrop is fine for this). 260/230 ratio is what you're looking for here, chiefly. Good RNA should have a 260/230 of 2.0 or greater. Lower than this and you're most likely looking at guanidium salt contamination. Trizol is a guanidium-isothiocyanate based RNA isolation method, so guanidium carryover is fairly common.

Given that guanidium salts are chaotropic, they're obviously incredibly inhibitory toward cDNA synthesis (or indeed any downstream enzyme-based treatment), so you want to get rid of them if at all possible.

I would be wary of using any RNA with a 260/230 lower than maybe 1.7-1.8, and would definitely prefer 2.0 if achievable, so if this turns out to be your problem, that's the benchmark to aim for.

Luckily, guanidium can be fairly easily removed (or at least, substantially lowered) by simply reprecipitating your RNA (either by ethanol or isopropanol precipitation).

As a further aside, the isopropanol precipitation suggested by the trizol protocol is very, VERY temperature sensitive: isopropanol precipitates RNA very well at room temperature, but cold isopropanol ALSO precipitates guanidium salt by the bucketload. Always make sure your isopropanol precipitations are carried out at room temperature.

Spec your RNA (nanodrop is fine for this). 260/230 ratio is what you're looking for here, chiefly. Good RNA should have a 260/230 of 2.0 or greater. Lower than this and you're most likely looking at guanidium salt contamination. Trizol is a guanidium-isothiocyanate based RNA isolation method, so guanidium carryover is fairly common.

Given that guanidium salts are chaotropic, they're obviously incredibly inhibitory toward cDNA synthesis (or indeed any downstream enzyme-based treatment), so you want to get rid of them if at all possible.

I would be wary of using any RNA with a 260/230 lower than maybe 1.7-1.8, and would definitely prefer 2.0 if achievable, so if this turns out to be your problem, that's the benchmark to aim for.

Luckily, guanidium can be fairly easily removed (or at least, substantially lowered) by simply reprecipitating your RNA (either by ethanol or isopropanol precipitation).

As a further aside, the isopropanol precipitation suggested by the trizol protocol is very, VERY temperature sensitive: isopropanol precipitates RNA very well at room temperature, but cold isopropanol ALSO precipitates guanidium salt by the bucketload. Always make sure your isopropanol precipitations are carried out at room temperature.

Dear John Hildyard

Thanks for your suggestion, I did it ( ethanol or isopropanol precipitation, 5 min, 12000g at 4 oC), but I didn't get good results. Can you please tell me  any other suggestion? With thanks.

a troubleshoot could be the room temperature of the lab!!

Dear Pietro Di Fazio

Thanks for your answer, but What do you mean from room temprature?? 10-15 oC ?

Ethanol precipitation protocol:

(use 2ml tubes)

Make RNA up to 500ul with DEPC-treated/RNAse free H2O. (can use smaller vols, but I find these volumes easier to work with)

Add 50ul (0.1vols) of 3M sodium acetate, pH 5.5

Add 10ug of glycogen (optional -improves yield)

Add 1.5ml (3 vols) ICE COLD ethanol.

Mix well

Store at -80 for 1 hour, or at -20 overnight.

Collect by centrifugation at 12-13k rpm in a chilled benchtop, 10mins, 4 degrees C.

Discard supnt, wash pellet 2x with ice cold 70% ethanol (can add this gently so as not to dislodge the pellet). 

Air dry 15mins, dissolve in appropriate vol of H2O.

Isopropanol precipitation protocol:

Make RNA up to 500ul with DEPC-treated/RNAse free H2O. (same as above, can use smaller vols if desired)

Add 50ul (0.1vols) of 3M sodium acetate, pH 5.5

Add 10ug of glycogen (optional -improves yield)

Add 500ul (1 vol) ROOM TEMPERATURE isopropanol

Mix well

Incubate at room temp (i.e. just leave them on the bench) for 20mins or so.

Collect by centrifugation at 12-13k rpm in a chilled benchtop, 10mins, 4 degrees C. (chilling at this point does not seem to significantly increase salt precip)

Discard supnt, wash pellet 2x with ice cold 70% ethanol (can add this gently so as not to dislodge the pellet).

Air dry 15mins, dissolve in appropriate vol of H2O.

Dear John Hildyard

Thanks a lot. 

Hi,

Whether integrity of your RNA extracted looks good on gels (maybe it could be good if you show it that here) and the ratio 260/280 and 260/230 given by nanodrop is good as well,  I think that you have RT inhibitors in your RNA. You could try to perform an RT reaction with RNA diluted perhaps 1:5, 1:10. Sometimes doing that your decrease the concentration of the inhibitor of RT and still is enough for cDNA synthesis. Anyway, you could remove most of inhibitors purifying RNA by RNeasy column from Qiagen (Once I used those column and I increased the ratios that I did mention above). Some technical issue, as John said be aware about isopropanol, always precipitate at room temperature (22-25C) 10-15min and after you perform centrifugation 15min at 4C 12000g as trizol protocol suggest. I perform one wash wih Etoh 75%. 5ug of Glycogen is largely enough to precipitate RNA, higher concentrations could inhibit RT reaction. Be careful about abundancy of your RNA target, perhaps before you were performing RT-PCR of high abundant RNAs and now it is low abundant RNA target or even worst maybe your new RNA target is very low or not expresses at all!. Good Luck!

Hi Reyhaneh,

I had a similar problem before and it turned out that too much ribosomal RNA was inhibiting the reverse transcription step. I never had a problem with qPCR when I extracted RNA from ~100,000 cells, but then when I started extracting from ~10M cells, my RT-PCR would fail because there was so much rRNA in my reverse transcription step. I ended up using the NEB polyA spin columns to remove all non-polyA RNA and immediately my RT-PCR started to work.

Alex

You should validate your RT-PCR machine by conducting a number of tests like selectivity, Trueness, False positive, False negative test. You have to run RT-PCR with spiking sample also. You can also try by changing extraction kit.

My protocol is slightly different:

1 - I plate 10(6) cells in each well for a 6-well plate

2 - let them grow and adhere overnight

(optional) right now you can treat cells with what you wish (ATP donor, LPS, sodium bissulfite, etc)

3 - then I use 500ul of trizol per well, each two wells make one sample (1ml)

4 - for each 1ml sample I use 200ul of chloroform, room temperature. vortex it strongly. rest for 3-5 mins at room temperature.

5 - spin at 15.000 rpm, 15 mins, 4C

6 - recover supernatant, discarding both the trizol and the interphase (which contains salts and dna) properly

7 - then I use 500ul of cold isopropanol (not ice cold, just cold ~10C), vortex a little bit.

8 - rest for 10 mins at room temperature

9 - spin at 15000 rpm, 15 mins, 4C

10 - you should now have a visible pellet, even though it may be a small one it's there

11 - then I wash this pellet using 70% cold ethanol, dilluted in DEPC water (you can use absolute grade if it pleases you and you don't want to spend too much DEPC water)

12 - spin again, for 10 mins, 15000 rpm, 4C

12 - discard ethanol gently and properly, retaining the pellet

13 - cover the table with soft tissues and let them air dry inverted

14 - the pellet should be super visible now. if it's not, then it's highly probable your 260/280 ratio and the concentration will be too low to analyze.

15 - dillute visible pellets with 30ul of DEPC water, or 10ul for invisible ones

16 - treat with DNASE I and EDTA (as per your usual protocol)

17 - quantify with nanodrop (I use GE nanovue plus)

18 - 260/280 ratios should be between 1.7 and 2.0 (optimum results are obtained when ratios are like this. I tend to proceed even if these are below 1.7, because sometimes it does amplify, but it's a just a shot). if you have enough samples, just discard the ones too far below 1.7 and keep the ones near 1.7 or above.

anotate concentrations, dillute them in DEPC water accordingly. At this point I like to vortex a little bit, then centrifuge. it helps getting the rna dissolved.

I adjust to a concentration of 150ug/ml, but this is up to your protocol.

you can store these samples in -80C indefinitely, and -20C for a couple of months. 

then use as desired. make sure you're running controls with every test plate.

RNeasy kits work best in my experience. Try diluting your RNA as this usually gets rid of any inhibitors. 

For now, you can "rescue" your samples by cleaning the cDNA with the Qiagen PCR purification kit. Diluting the cDNA usually works as well. I once had a standard curve, where the undiluted and 1:10 performed worse than the 1:100, 1:1000 etc. That is because the contamination is diluted, too. You could run an RNA cleanup protocol (Qiagen) for cleaning your For future, Make sure you don't use more than 100 mg tissue per 1 ml of Trizol, make sure your homogenization is 100% - when these things are not good you will get contamination from the tissue. Then, after the first chloroform step I personally do another chloroform step with the aqueous phase, so that I get rid of any left-over guadinium salts (mentioned above). I do isopropanol precipitation by centrifuging for 1 h at 4 degrees. It's much more efficient than the standard protocol. Make sure your RNA pellet is thoroughly dried, any left-over ethanol can inhibit the PCR, too. The above suggestions with the 260/280 are of course also important.