I plotted Ct vs log conc. of my standards and the slope of the curve is -1.79, giving me an efficiency of 262%.
It depends on whether your first few standards were too concentrated thus inhibiting the qPCR at those dilutions (giving higher Cq values than are reasonable there) and bending the curve downward exaggerating the efficiency above 100%... also depends on (if you are using a SYBR Green approach) whether or not you have primer dimers and/or other non-specific side reactions occurring that also contribute erroneously to overall signal and result in again flattening your curve at the least concentrated sample points - keeping the curve falsely flattened and thus exaggerating the efficiency again. If both are happening, then you would be getting this nightmare two-fold (or more...)...
It depends on whether your first few standards were too concentrated thus inhibiting the qPCR at those dilutions (giving higher Cq values than are reasonable there) and bending the curve downward exaggerating the efficiency above 100%... also depends on (if you are using a SYBR Green approach) whether or not you have primer dimers and/or other non-specific side reactions occurring that also contribute erroneously to overall signal and result in again flattening your curve at the least concentrated sample points - keeping the curve falsely flattened and thus exaggerating the efficiency again. If both are happening, then you would be getting this nightmare two-fold (or more...)...
Could it be because of inhibitors and very low starting material? I am using sorted cells and only have enough for duplicates in the reaction. I checked the efficiencies of every pcr reaction I ran and on average the efficiencies of my housekeeping gene are 278% (slope -1.73) and 272% (slope -1.75). The efficiencies of my test gene are 796% (slope -1.05) and 877% (slope -1.01) !!!! I am using Taqman primers. my 260/280 ratios for RNA vary from 1 to 2 and on average they are 1.4-1.5.
Justine, for RNA, the ideal 260/280 ratio is between 1.8-2.1. If checking on proteins, this ratio of 1.5 indicates usually 90% protein! 70% RNA should show at least 1.94 coefficient, as ratios are not reciprocal due to different extinction coefficients of nucleic acids and proteins, respectively.
Try using only good preps in order to make a solid calibration curve, do not mix them and do not average anything. Low qual preps could be further purified by cold (even at -20 C) precipitation (isopropanol 3v to 4 v RNA solution), centrifugation at high speed, 70% ethanol wash, and resuspension to a suitable volume to reach whatever concentration you need.
If you don't have enough sample material, try using a lysis method to get RNA rather than extracting it by no matter which protocol. There is a good kit from Roche which is called Real TimeReady cell lysis (the flyer attached) or equivalent from Life Technologies called "cell to Ct". You will be able to get RNA suitable for fine qPCR from as low as 3 cells. In 5 minutes!
I will start with RNA quality improving in the first place.
About your efficiency - every efficiency above 100-110% indicates lots of unspecific products which give you fluorescence ("amplification") but out of your scope, i.e. the specific product. If you have possibility, run a gel to check that. TaqMan primers? I think you mean TaqMan probe, right? Your efficiency should be closed to 100, preferably not exceeding it, R2 closed to 1, ideal slope -3.32.
Run a gel and see what you've got.
Hi Justine,
More details would be needed. How many cells are you extracting for RNA? How do you extract the RNA? Do you test the RNA integrity before use? Are you using a one-step qPCR kit? If not, how do you prepare your cDNA? If you prepare cDNA, how much RNA do you add per RT rxn? What size are these RT rxns? Are you being certain to add the same amount of RNA to each RT reaction? How much sample do you add per qPCR? How large is each qPCR reaction? What mastermix do you use? What are your thermocycling conditions? What are the Tm values of your primers and hydrolysis (Taqman probes)? How specific are your primers and probes for their respective targets? Do you DNase treat your RNA at some point? Do your Taqman probes span an exon-exon junction? What species are you working with? Providing these details would help us to help answer better.
Does your melt curve indicate more than one amplicon? Have you physically observed your amplicon on a gel? I would expect you are generating more than one product.
Hi everyone,
Thank you for all your insights...I am using sorted subsets of human B lymphocytes. The number of cells I get ranges from 10,000 to 1 million. I extract the RNA with Trizol and further purify it with the Applied Biosystems Picopure RNA isolation kit. I do a DNase step in there. I measure the RNA on the Nanodrop to be certain there is RNA there and to examine the 260/280 ratios. I don't fully trust the RNA concentration Nanodrop gives me and am more interested in the purity. On average my 260/280 ratios are worse on samples that came from fewer cells. After this I proceed to reverse transcription and I use the IScript cDNA synthesis kit. I follow the instructions exactly and then store the cDNA in the -20C freezer until qPCR.
*There were times I had to leave the RNA on ice for 1 hour after elution but before reverse transcription, either because someone was using the machine and I had to wait or I had to go to lab meeting or class etc.. Could that be affecting the integrity of the RNA? This is one thing I am worried about.
Now for qPCR, I use a Taqman assay pre designed from Life Technologies. The assay does span exons, and for the master mix I use the Light Cycler 480 Probes Master mix. I don't do a melting curve because I'm not using SYBR green. I also don't add the same amount of RNA to each RT reaction, I reverse transcribe everything. But for qPCR, when I am comparing the housekeeping gene to test gene both conditions have the same amount of cDNA.
Thermocycling conditions:
Pre-incubation 45C 5min 95C 3min (1x)
Amplification 95C 15sec 60C 45 sec (45x)
Cooling 70C 2min 37C forever (1x)
Each reaction well has a final vol. of 12ul
Donita, that kit looks like it would be very suitable for my situation. would I be able to use the Real Time Ready lysis kit with the BioRad IScript cDNA synthesis kit?
Your setup looks fine. That high efficiency is very strange to me for a probe assay. Do you see similar problems with other genes?
Starting material is important and 10K bcells is a challenge, but you should be fine with more than 200K. I recommend to use 1ml Triziol independent of your cell count, you would be able to get more from the upper phase. If your RNA is somehow degraded, you may loose degraded fragments during RNA claen up. Have you ever tried directly using RNA isolated with Trizol? How much do you dilute your cDNA after reverse transcription? ABI pimers works without problem in most cases, you should avoid thaw-freeze cycles, direct light and storage.
Perhaps you mentioned it but are you obtaining an RNA integrity number (RIN)?
It has been my impression that in order for publication this analysis is required for credibility of your analysis.
Chris, what is an RNA integrity number? Are you referring to the 260/280 ratio? I have those measurements for each sample...
Hi Everyone,
I have done an experiment testing my cDNA I use for standard curves with a different housekeeping gene. For this gene, I got an efficiency of 97% (slope -3.39). But it seems that, no matter what, the efficiency of my gene of interest will always be greater than 100%.
Which leads me to another question: If my efficiencies for each gene are consistent each time I do the qPCR is this ok? For example, I tested 4 samples one day and got efficiencies of 78.6% for housekeeping and 160.4% for gene of interest. Another day I did the same experiment with 2 more samples and got efficiencies of 77% for housekeeping and 168.9% for gene of interest. Some people in the lab told me as long as the efficiencies are consistent each day I do the experiment, even if they are consistently crappy that my results should be fine. I think they've been consistent: 77% and 78.6% is pretty much the same and 160.4% and 168.9% are within 10% of eachother.
But I am also wondering if I should go ahead and re-analyze using the housekeeping gene with the 97% efficiency anyway? It seems like no matter what I won't be able to improve the efficiency of my gene of interest. Thoughts?
The RIN number can be generated by a special analyzer... here is a reference that might help introduce this :
RNA integrity and the effect on the real-time qRT-PCR performance
Simone Fleigea,
Michael W. Pfaffl
Dear Justine, I cannot confirm that RTr cell lysis kit works with iScript from Bio-Rad. Our customers generally do not mix reagents from different suppliers, as they are perfectly satisfied with Roche reagents. What I can point out for you [as LifeTech reagents are common to you] is that there is a publication using Cell-to-Ct in conjunction with iScript. I attach the paper under question. But maybe this was not a good idea, as all direct lysis methods are referring to cultivated cells...
Concerning your isolation method, I don't understand why you use TRIzol before the LifeTech PicoPure RNA Isolation Kit which is designed - according to them - to recover total RNA from fewer than ten cells. Directly, I mean. If you have minimum 10,000 cells this is definitely much more than fewer. It should work as is, without any TRIzol, applying the whole protocol as indicated in the manual.
Leaving the RNA eluates 1 h before RT should not affect the integrity that much to a complete failure. I used to work with RNAs from viruses for 18 years, in even worst conditions. I never failed due to that. I fear most your isolation method which is kind of weird.
I think similarly to Korcan, at least try directly using RNA isolated with TRIzol. Give up mixing up unnecessary isolation methods, use one or another.
Instruments and kits able to help checking RIN are kind of recent for me. I did use Bio-Rad's machine, but if you don't have it already, it will be difficult to get one in due time. You can do a simple denaturing gel electrophoresis for that, but... please consider allocating enough time to be there to finish your experiment. To get minimum clear information on what you should look for, please visit for example this page, go to tab Assessment of RNA Quality and read http://www.thermoscientificbio.com/rna-electrophoresis/#assessment_rna_quality
About iScript, it is worth to note that too much RNA suffocates the RT reaction, so if you get more than 1 μg from your isolation the reaction should be scaled up for better RT efficiency first.
Last, as Roche expert I question your PCR protocol. I do not see the reason why pre-incubating the reaction at 45 C for 5 minutes? Why only 3 minutes at 95 as long as the LC480 probes master is a hot-start which requires minimum 5 min for activation? Most probably, you do not activate your qPCR properly! Amplification 95 C 15sec 60 C 45 sec (45x) - where is your 1 sec extension step? This is a TaqMan assay you've said, do you expect polymerase to work at 60 C and cleave your probe? For 2-step annealing (and primers) should melt at higher temperatures, isn't it.
Cooling 70 C 2 min 37 C forever (1x) - again, strange for me, whichever instrument you are using.
Again, if I were you, I would check steps from the beginning, ONE-BY-ONE. Try respecting the intended use of your reagents and the recommended protocols.
Doinita,
I have tried using trizol in the beginning without the PicoPure kit. I didn't like it because I had highly impure RNA. My colleage who is one of the senior scientists recommended using Trizol in tandem with the Picopure kit because it works well for him and since then several of us have been using his method. I have tried extracting RNA without Trizol once using the kit but that turned out to be a disaster and I haven't done it again since.
The RIN thing is new to me too and I don't think we even have that kind of instrument in the building. For the reverse transcription, I made sure I scaled up the reactions properly when I had to.
For the PCR protocol, I also wondered whether I should have added in a 72C synthesis step for each amplification cycle, but read that Taqman assays with shorter amplicons
I FIXED THE PROBLEM! Lengthening the activation of the polymerase to 10min really worked. Thanks for the suggestion. I now have efficiencies of 98% and 106%! I also deleted the 45C step and the 70C cooling step.
I'll accept values ranging from 90-110% from now on.
- Badges
- Science topic