I am working with adipose tissues. I normally make cDNA using 'High capacity cDNA RT kit' (Thermofisher). For making standard, I dilute my cDNA by 10 fold with Tris HCL buffer which is my arbitrary conc, 10,000. Then I serial dilute it for 5000, 2000, 1000 and 100 arbitrary conc. I make a standard curve by putting 5 ul of diluted sample in well with Sybr green and primer. My samples are diluted by 25 fold with Tris buffer. I normally used to get good efficiency for housekeeping genes and my target genes in qPCR.
However, now I have some genes which are expressed at low level (Ct> 30) and the standard curves and melt curves are not looking good. I followed a post here where a person was experiencing similar problems. From that post, I decided to concentrate my cDNA by taking 3 ug instead of 1 ug of mRNA and reverse transcribed it using Superscript IV reverse transcription kit (Thermofisher) as this kit claims to reverse transcribe upto 5 ug of RNA. Weirdly, after the reverse transcription, my 260/280 values were ok but the conc did not increase to my expected level (previously I used to get 1.1 ug/ul using 1 ug of RNA but now using this kit, I am getting 1.48 ug/ul). I still can accept that but my 260/230 ratios are getting lower (1.43 to 1.77) in cDNA. I believe the contaminants are also increasing in this process. I am planning to dilute my standard for qRT-PCR by 2 fold only instead of 10 fold. And I want to dilute my sample by 5 fold instead of 25 fold. Do you think that it is going to work as I am diluting less?
Has anyone ever suffered with this problem? Please give advice me for quantifying low expressed genes by SyBR Green method.
From my own experience, ratio of 260/230 are usually low when you have low concentration of RNA available. That does not necessarily means you have any sort of contamination. Having said that, make sure you do your clean up before any further analysis.
Thanks for your reply. But my 260/230 values are low for cDNA and after reverse transcription, I dilute cDNA and use it in qRT-PCR. There is no clean up step from final cDNA to qRT-PCR. I have already cleaned up my mRNA. Does anyone have suggestions on how to quantify low expressed genes in qRT-PCR. Is concentrating cDNA is a good method?
In addition, low 260/230 ratios induced by guanidine thiocyanate don't disrupt downstream downstream applications at concentrations up to 100 mM.
https://www.qiagen.com/gb/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en
Sometimes, low 260/230 ratio also depends on the way you extracted your RNA. I always get low 260/230 ratios with TRIZol-extracted RNA. It's most likely to do with high salt concentrations, which isopropanol precipitation is not so effective at removing.
Agree with @Faraz Khan. A 260/230 value just indicates reagent contamination and especially prominent when you start with very small tissue amounts but still using same reagent volumes. You should focus more on 260/280 values. In my own experience, I have worked with RNA with 260/230 around 1 which showed no degradation and even acceptable RIN values in Bioanalyzer. I have used these samples for microarray as Bioanalyzer results were satisfactory.
For me, I have tried 1.5 ug RNA for cDNA synthesis but kept all the reagent volumes same except the SS III RT (1.5 times more). You can never quantify cDNA with Nanodrop or gel. However, you can try using Pico-Green dye.
I usually, deal with genes having low expression like ethylene-regulated transcription factor genes in plants with Ct around 35. I have also increased the number of cycles to 45 instead of 40 in the RT-PCR reaction protocol.
Thanks a lot Debatosh. Have you eliminated genomic DNA contamination from your RNA sample before reverse transcription? If you have, then which method did you use? Can you elaborate a little bit?
Afsana Trini, I use TOYOBO One-step RT-PCR Kit,one reagent there name gDNA remover is used to remove the genome DNA.I guess it's actually a RNAase free DNAase.
But some of my collegue usually design primers that is to amplify the region spand the exon-exon to avoid amplifying the intron region of the genome DNA.
Thanks Qing. My primers are also designed to span the exon-exon junction. I will have to remove the genomic contamination and observe how it improves my Ct values and melt curves.
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