Hello every body.
Using (SensiFast Sybr No-ROX one step kit) from Bioline, I did qPCR experiment used one -same- sample with 3 different concentrations of RNA and two different reference genes. Concentrations used 300 ng in 20ul reaction, 400ng and 500 ng.
The reaction in wells A, B and C in the attached images included the master mix according to the procedure and the steps as mentioned in the kit.
D and E include the same sample of concentrations (300ng and 500 ng) the difference is that the RNA was treated with 2ul DNA-wipe for 2 minutes before adding the Kit master mix.
My lab mate used the same samples I used in this experiment, but she used the 2 steps kit (prepared cDNA and then qPCR) and she got nice melting curve!
Can you please discuss with me this issue? all suggestion will be appreciated.
Thank you in advance
Hi Helmi. My initial impression is that yes, it looks a bit of a mess. But before I say more could you confirm that columns 1 and 2 are replicates (or are these the two reference genes with no replicates), and that A, B, and C represent the 300,400 and 500ng reactions respectively.
Hi Roger,
Thanks for your response
the columns 1 & 2 are two different reference genes one is GUSB and the other is HPRT. And I used the same template for both genes.
Yes A, B & C c represents the the concentrations 300ng , 400ng and 500ng respectively.
Thanks Helmi. Yes the results are completely messy and not useful. I cannot work out what went wrong, because very little makes any sense. The lack of replication also does not help. Some things that don't make sense:
- Moving from Row A to C the Cq should be about 1 cycle less, for both reference genes. This works for column 2, but in column 1 it is about 12 cycles more.
- The position of the melt peaks for column 2 are so variable, that it looks like the same product is not been made in all samples. Usually I would expect melt peaks in the mid to low 70s to be caused by primer-dimers.
- Treatment with DNAase should make the Cq increase if anything, but it reduces a lot.
I suggest for a start that you try getting the same result as your lab mate, using the same two step kit. Also consider getting the PCR working using a gDNA preparation, then move onto cDNA - assuming the intron-exon aspect is not a problem for your primers. Or, use cDNA made with two step kit, then put it into your PCR, and make sure it is working appropriately. Generally I would recommend you use the two step method in this application.
Best regards
Roger Latham
Dear Helmi
I think Roger makes some excellent specific points so I will try and illuminate the problem of multiple melt peaks with a few general choice comments:
1. I too would use the 2 step approach and make total RNA followed by cDNA
2. Having made cDNA I tend to validate initially by standard agarose gel based PCR (that is RT PCR) using a housekeeper gene like GapdH
3. Non specific bands on gels mirrored by multiple melt curve peaks are or can be non specific products and/or primer dimers; both aspects of poor primer design and/or inappropriate annealing temp
4. Thus, having designed primers to exon-exon boundaries using primer Blast and made sure the possibility of putative primer dimer formation is minimal I tend to perform a set of annealing gradient reactions from Tm-3C to Tm +2C in 1C intervals; i.e. 6 x separate PCR reactions from Tm-3C; Tm-2C ....Tm+2 C. I then run these products on an agarose gel; select the annealing temp that yields the most specific (single) band in the largest amount (highest PCR efficiency) and then carry that same optimal annealing temp over into my actual qPCR reaction
5. If non specific bands are evinced by melt curves, to circumvent one possibility of secondary specific reactions I will always, having designed primers, confirm their specificity by BLAST
6. Finally, I will add that even if I DNASe treat samples I will also specifically target cDNA by designing exon exon boundary primers using primer Blast or taking the ref sequence from NCBI primer blast and designing primers using IDT primer quest
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
https://www.idtdna.com/Primerquest/Home/Index
I will make one specific point about DNAse I treatment and Cq/t values
Unless you use a DNAse I that is certified as RNase free, e.g. rDNAse I (turbo) from PE or Qiagens RNAse free DNase I is is not uncommon for treatment with DNase I to cause some breakdown of RNA as well. This degradation reduces the efficiency of your RT reaction and therefore increases the Ct/q value during qPCR
Thanks Roger and Laurence for your valuable comments.
I repeated the experiment and the results were very good I got a single melting curve beak.
I fixed the concentration to 400ng in the 20uL reaction and it worked very good and I repeated the experiment again with 200ng in 10uL reaction and it was very good and consistent.
Dear Dr. Helmi Y. Alfarra
i suggest you read this
Interpreting melt curves: An indicator, not a diagnosis
http://www.science.smith.edu/cmbs/wp-content/uploads/sites/36/2015/09/Interpreting-melt-curves.pdf
Melting Curve Analysis
https://www.fluidigm.com/binaries/content/documents/fluidigm/resources/melting-curve-analysis-ug-68000118/melting-curve-analysis-ug-68000118/fluidigm%3Afile
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