I am currently performing qPCR work on intestinal (duodenum and jejunum) tissue collected from the pig small intestine and stored at -20°C in RNALater Stabilization Solution until qPCR analysis. There is high variability in my data (Ct values) for both reference and target genes between biological replicates (pigs in same treatment group) and across treatment groups. However, all technical replicates (same cDNA sample plated in duplicate) are within 0.5 cycle of each other. All melting peaks are perfect as well. Primer amplification efficiencies are all within 90-110% (96% for RPLP0 references gene). RNA A260/280 ratios are all within 1.8-2.1. I have extract fresh RNA recently but the results did not change. There is no stability with the Ct values for the reference gene between biological replicates in the same group and across treatment groups, while the standard deviation for technical replicates is acceptable. I have also tried RPLP1 and B2M as reference genes but they also follow a similar trend. Target genes also follow this trend.
The following are the kits/protocols used throughout the entire process;
- RNA extraction protocol (Using RNeasy Mini Kit from Qiagen)
- cDNA synthesis protocol (Using SensiFAST cDNA Synthesis Kit)
- PCR protocol (Using LightCycler® 480 SYBR Green I Master Kit)
Maybe the high variability between different pigs within the same treatment group is due to natural biological variation (gender - both male and female in study, circadian rhythm, menstrual cycle etc.) I am not sure? Although, this does not explain the high variation with the reference gene. I know the reference gene is meant to stay stable irrespective of treatment type. Thanks,
Cathal
Dear Cathal Dold,
The variation in Ct values means variation in the amplification step, which could be due to either:
- Technical error (loading samples and reagents into the PCR plate, variation in the step of cDNA preparation "using more RNA than recommended or variation in the RNA concentrations used", instability in the RNA concentration evaluation (nanodrop?!), or different replicates have different RNA quality "good quality RNA molecules are not degraded", you may check the RNA quality by running on agarose gel).
- Biological variation (you already mentioned this possibility), you might know that not all reference genes are stable for the different treatments, so you might consider trying different reference genes and find out which one is more stable following your treatment (you can use RefFinder for this purpose [https://www.heartcure.com.au/reffinder/?type=reference])
Good luck!
Yassouf
Hello, Cathal. I would suggest to perform a validation experiment for all of your reference genes. This means that you should take all your samples (control as well as experimental), perform cDNA synthesis from the same amount of RNA from each sample and subsequently take the same amount of cDNA into qPCR (preferably all genes at the same time). All Ct values received you should analyse using the reffinder tool: https://www.heartcure.com.au/reffinder/. This should answer your question whether your reference genes are truly stable or you should search for another set of references genes. For reference, please, see Article Housekeeping Genes for Parkinson’s Disease in Humans and Mice
. Best regards.
Those data are entirely expected. The entire point of including a housekeeping reference gene and a standard curve is to be able to calculate expression relative to the housekeeping gene and allow to you compare between samples.
The variation is most likely due to differences in how much tissue you started with.
Good luck!
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