Possibly silly question: following cDNA synthesis, do you 'clean up' your samples using a column kit or just dilute the cDNA and use it straight away? There's arguments for both methods but if you use a kit then I am wondering if there's EDTA in the elution buffer.

Artur

Yes, thank you, I meant the primers amplify regions between 100-300.

John

I chose a 1:4 dilution because I imagined 1:10 becoming too dilute from my initial concentration that it might not be accurate at lower concentrations. Let me illustrate.

                    1        2            3              4             5              6              7

1:4            150    37.5    9.375    2.3437    0.5859   0.1464    0.0366

Ct @ 1:4  25.5   25.5     25.5        25.5          26           28          29.9

I have rounded these Ct numbers off, but this is about what I am getting for my samples. My cDNA kit gives me a maximum of 250 ng/µL (Clonetech ecodry premix). So my initial dilution was not even 1:2 because I was trying to get concentrations for the lower 20s. What kit do you use and what is your initial concentration?

I could use more µL into the PCR reaction instead of water to increase the spread, but the problem is those higher concentrations.

The fifth dilution of 1:10 would be less than the seventh here, going into the 30s for Ct values.  The log base 2 of 150 and 37.5 are 7.228 and 5.228 respectively, meaning I should be seeing 2 Ct values between these dilutions. I should be seeing my first dilution around 18 cycles, 2nd at 20, 3rd at 22, 4th at 24.  Once it gets to the 5th, the values start to shape up but the first dilutions are clustered around 25.5.

I will try the DNAse step this morning.  I diluted my RNA samples by the same factors as I have to get to cDNA so both should have the same gDNA concentration.  The genomic DNA got a Ct value at ~30.  Perhaps the concentration of DNA itself is acting as an inhibitor.

As for the melt curve, I used that to determine if there was secondary structure or primer dimers, as suggested by a trouble shooting guide.

John,

Ok, so your invitrogen kit was 5µg in 20 µL, ending with 250 ng/µL.  A 1:10 dilution reducing it to 25 ng/µL. If your starting concentration used 50 ng, then you used 2 µL per reaction.

That is between my first and second dilution. I will follow your advice on 1:10 dilutions, though I am afraid the first three might cluster as they fall in the range of those I pointed out before for my 1:4 dilution.  

I will also do a DNAse step and check for EDTA in reagents.  Thanks for your advice.  I'll post whether this fixes my problem.

Most RT-reactions (cDNA) need to be diluted at least 10 times before qPCR to dilute out the inhibitors that are present in the RT reagentia. If you dilute them not enough, the amplification curves are affected by it and the responding measurements are not on the rest of the standard curve. Your initial dilution of 1:2 is not a good first standard.

I read in this thread an error that a lot of people seem to use: if you add 250 ng/µL RNA in a RT-reaction, the resulting cDNA concentration is not also 250 ng/µL. The resulting amount of cDNA depends on a number of factors, like the amount of template, the priming method used (oligo-dT priming primes only mRNA, which is ~2% of total RNA) and the efficiency of the RT-reaction which is never 100%. So basically you can’t know the concentration of cDNA from this information.

That said, I think 5 µg of RNA in a RT-reaction is a lot, did you try to use less RNA? Did you ever do an optimization?  If you titrate RNA into a RT-reaction, there might be a plateau where adding more RNA only has a negative effect downstream of your procedure (I did this experiments myself with different RT kits).

By the way, did you use a 3-step PCR program? If yes, how long is the synthesis step? (you mentioned only the annealing step).

No DNase: is not the right way for gene expression studies as experienced myself. On the other hand, I found potential RNA hydrolysis in some samples after DNase treatment. Having such experience, I applied RNase inhibitor during DNase treatment to stop RNA hydrolysis, and that worked out an excellent way of removing genomic DNA from the RNA sample. Now come to the point: if we do not remove genomic DNA, in what degree it can influence mRNA expression? I found a significant level of changes in mRNA transcripts level even in the control samples, meaning that even though getting distinct bands for 28S and 18S rRNA with no smears on agarose gel electrophoresis, it doesn't provide the assurance of NO Genomic DNA contamination in total RNA. Check Mg2+ and EDTA in your reaction.

In the case of cDNA concentration in Reverse Transcription, it is better to use 12.5 ng/uL cDNA rather than putting your time for adjusting cDNA dilution.

Marco-

Thanks for your advice.

The first time I tried this experiment, I did not assume a 1:1 conversion from RNA to cDNA.  I actually tried to quantify the cDNA and it gave me very high numbers.  In response to this, I diluted the cDNA too much and could not get amplification until after 30 cycles. I did not know what I did wrong for days until I found a conversation on the internet.  It said to assume a 1:1 conversion and quantifying the cDNA won't work because of the left over reagents.

But you do bring up a good point, maybe the amount of RNA included needs to be optimized.

I did have two sets of RNA- shoot RNA with 5µg added and  root RNA with 3µg added. This morning I checked results from the root RNA.  I followed the advice of John and did 1:10 dilutions. I am adding the excel file of what the results were.  But the summary is this:

Primer 6 196.3% efficient, R^2 0.994, SLOPE -2.12

Primer 7 198.7% efficient, R^2 0.98.7, SLOPE -2.04

Primer 8 214.0% efficient, R^2 0.990 SLOPE -2.011

Primer 9 189.9% efficient, R^2 0.984 SLOPE -2.163

I followed a 2 step PCR program as instructed by the taq mix protocol.  I had been using SsoAdvanced Sybr Green, but yesterday I used SsoFast.  Both instructions use melting followed by annealing and extension together in one step.  

I have more tissue to extract today and I may go easy on the amount of tissue- maybe that will help reduce final concentration of inhibitors as well.

Hossain-

Yesterday I tried the zymo clean and concentrator -5 for RNA clean up with DNase I.  Afterwards I had quantified the original sample with the clean sample.  The RNA is used was a bit degraded because I have had to pull out of the freezer more often than I had expected.  Still, original shoot RNA quantified at 312 ng/µL in 36 µL but the treated version finished at 66 ng/µL in 15µL.  This has happened to me before.  This time it means started at 11,232 ng of RNA and ended with 990 ng.  That is a loss of 91% of my RNA when trying to clean and concentrate it.  

Instead, I am diluting my RNA by the same factor as I have to transform into cDNA and then for qPCR dilutions.  So if there was any genomic DNA contamination carried over in cDNA samples, it will exist in the same concentrations as the diluted RNA samples.  I then run both together in qPCR to determine if genomic amplification is occurring with the cDNA  samples.  

Hello Michael,

I also work with plants (Arabidopsis) so maybe I can help you somehow. 

Normally I isolate RNA using Trizol reagent, then I treat the samples with DNase I from Roche. This step is crucial because otherwise you have contamination of gDNA although you can not detect it on agarose gels. You should use RNase inhibitors when treating the samples with DNase I to avoid RNA degradation. Then you inactivate DNase I and extract the sample again (you can follow Roche protocol).

Once you have the RNA you can check the quality on a gel and quantify it. For the RT reaction I use SuperScript 3 from Invitrogen. Normally I use 1 µg of RNA in a 10 µL reaction or 2 µg of RNA in a 20 µL reaction, depending on how many primers I need to test. After the RT reaction I treat with RNase H, because for some targets is important to avoid secondary structures.

I am using GoTaq reaction kit for the qPCR (Promega). I dilute my samples 1/3 and for the standards I usually do 5 dilutions 1/4 each. However for some primers I have to use other dilutions, for instance, when a transcript is present in low amounts in the sample.

I hope this information can help you. Do you have this problems with primers for housekeeping genes also?

Good luck!

Zaida.

Oh! I forgot. When doing RT-qPCR I always include the RT+ reaction (with enzymes) and the RT- reaction (without enzymes) because even after treatment with DNase I, sometimes you have some gDNA contamination. You should include this control specially if your primers are not intron spanning.