In my qPCR reactions the housekeeper ct values for GAPDH are very high (ranging from 22-39) and Actb values are undefined when I used the cDNA extracted from cardiomyocytes. When using control cDNA the values are normal.
Should I expect relatively higher ct values for housekeepers with cardiomyocyte cDNA or is the difference down to the cDNA quality (cDNA synthesized with low quantity RNA and RNA with low 260/230).
Any ideas would be greatly appreciated!
Thanks
Laurence Stuart Dawkins-Hall
Popular answer In my experience, the Ct values for housekeeping genes using HCM cDNA are typically around 20; in common in fact with Ct values in other contexts Thus, Ct values of 22 would not be unusual but above 25 and certainly up to 39 suggests low cDNA efficiency culminating in low copy number, perhaps due to low quality and/or quality of starting material To reiterate my previous answer: https://www.researchgate.net/post/Good_protocols_for_qPCR_of_cDNA_from_cardiomyocytes#view=586bbfce615e2779fb50ca04Make cDNA from >/= 50ng total RNA derived from cardiomyocytes and using superscript III or preferably IV; random primers and oligo dT; and perform RT @ 50C for 1-2 hours as detailed in the method attached to this answer but also your associated question specifically about RT These are the kits I would recommend using the SOP attached: https://www.thermofisher.com/order/catalog/product/18091050 https://www.thermofisher.com/order/catalog/product/4368814 To focus now on the downstream qPCR analysis, apart from RT as described I would also: Keep your amplicons short; about 100-150bp, preferably nearer to 100bp. This will increase the efficiency of your PCR cycling parameters Just as crucially design your 100bp spanning primers to within 1kb of the 3' end of your message: Although random hexamers facilitate random priming and hence qPCR upstream of this region, degradation of RNA occurs from the 5' end and thus your will have more efficinct and consistent priming and more accurate and robust PCR if you situate your primers within 1kb of the Poly A tail of your GOI In addition, when performing qPCR on cardiomycte cDNA as described I routinely add 5% DMSO to the sybr green qPCR reaction mix and also tailor the annealing temp to suite that of the primers as verified by cold standard PCR; In particular, make sure you can obtain a specific band on a gel from 1ul of your RT reaction derived from >/= 50ng total RNA and ensure that your primer Tms are ~ 65C and elect to use an annealing/extension step of Tm-2C; therefore 60-65C Finally, to ensure that your low 260/230 ratio is due to low concentration rather than GITC derived from the lysis buffer you could think about the way you purify RNA from cardiomyocytes; In particular: Try and use an RNA purification kit like RNEasy for low quantities of starting material (as opposed to trizol) When performing the wash step on the column let the ethanol based wash buffer sit for 1 min before spinning through and then repeat this wash: This more rigorous desalting will lower any GITC (lysis buffer) salt contamination and increase your 260/230 ratio In addition, elute with 30ul of molecular grade water pre heated to 37C: Add to the column; let sit for 5 min; spin elute and then add back to the column with an additional 20ul of water; let sit again for 5 min then spin through (the extra 20ul of water is designed to ensure that the elutant vol does not fall much below 30ul and especially 20ul as this results in poor elution, exacerbating issues with poor recovery from low amounts of starting material To increase the concentration of your total RNA you can precipitate with 3 x volumes of Ethanol @ -80C for 1 hour to overnight 1/10 vol 3M sodium acetate pH 4-5 And CRUCIALLY 1ul of 1mg/ml to 20mg/ml glycogen. The addition of glycogen will greatly enhance your RNA recovery Then, Make cDNA as described sing 1ul of this cDNA mix and then ensure your 3' biased primers to housekeeping genes and GOI, designed to amplify products approximating to 100bp, are exon spanning: This means you specifically target mRNA and not genomic DNA and thus avoid the need to digest with DNASE I to eradicate genomic DNA which can further degrade your RNA and reduce RT efficiency Use 'primer BLAST' in NCBI to make such exon spanning primers: https://www.ncbi.nlm.nih.gov/tools/primer-blast/ These measures should hopefully ensure the Ct of your housekeepers approximates to 20 and that your GOI are 20-30 (ideal range) in concert with a higher concentration of cDNA if you are using a dilution of your cDNA for cardiomyocyte qPCR: I typically use 1ul of the initial 1:5 diluted cDNA reaction mix I should also add Alisha that for cardiomycocytes GAPDH is not your best housekeeper. I deally you should nortmalise for for final analysis with 3 x housekeepers - but that is a whole other subject - and when performing qPCR on HCM, I typically use (and the literature suggests)
Dear Alisha
I have just answered another question about prior cDNA synthesis from cardiomyocyte RNA. I have done this myself in order to analyse gene expression in cardiomyocytes by qPCR
To answer this question:
B Actin
B Tubulin
B2 Micro globulin for example
I hope this helps in conjunction with my previous answer
3 votes
0 thanks
Saddam Hussein Abbas Baba
Hi Alisha
Check your concentration your primers and cDNA!!!
0 votes
0 thanks
Laurence Stuart Dawkins-Hall
In my experience, the Ct values for housekeeping genes using HCM cDNA are typically around 20; in common in fact with Ct values in other contexts Thus, Ct values of 22 would not be unusual but above 25 and certainly up to 39 suggests low cDNA efficiency culminating in low copy number, perhaps due to low quality and/or quality of starting material To reiterate my previous answer: https://www.researchgate.net/post/Good_protocols_for_qPCR_of_cDNA_from_cardiomyocytes#view=586bbfce615e2779fb50ca04Make cDNA from >/= 50ng total RNA derived from cardiomyocytes and using superscript III or preferably IV; random primers and oligo dT; and perform RT @ 50C for 1-2 hours as detailed in the method attached to this answer but also your associated question specifically about RT These are the kits I would recommend using the SOP attached: https://www.thermofisher.com/order/catalog/product/18091050 https://www.thermofisher.com/order/catalog/product/4368814 To focus now on the downstream qPCR analysis, apart from RT as described I would also: Keep your amplicons short; about 100-150bp, preferably nearer to 100bp. This will increase the efficiency of your PCR cycling parameters Just as crucially design your 100bp spanning primers to within 1kb of the 3' end of your message: Although random hexamers facilitate random priming and hence qPCR upstream of this region, degradation of RNA occurs from the 5' end and thus your will have more efficinct and consistent priming and more accurate and robust PCR if you situate your primers within 1kb of the Poly A tail of your GOI In addition, when performing qPCR on cardiomycte cDNA as described I routinely add 5% DMSO to the sybr green qPCR reaction mix and also tailor the annealing temp to suite that of the primers as verified by cold standard PCR; In particular, make sure you can obtain a specific band on a gel from 1ul of your RT reaction derived from >/= 50ng total RNA and ensure that your primer Tms are ~ 65C and elect to use an annealing/extension step of Tm-2C; therefore 60-65C Finally, to ensure that your low 260/230 ratio is due to low concentration rather than GITC derived from the lysis buffer you could think about the way you purify RNA from cardiomyocytes; In particular: Try and use an RNA purification kit like RNEasy for low quantities of starting material (as opposed to trizol) When performing the wash step on the column let the ethanol based wash buffer sit for 1 min before spinning through and then repeat this wash: This more rigorous desalting will lower any GITC (lysis buffer) salt contamination and increase your 260/230 ratio In addition, elute with 30ul of molecular grade water pre heated to 37C: Add to the column; let sit for 5 min; spin elute and then add back to the column with an additional 20ul of water; let sit again for 5 min then spin through (the extra 20ul of water is designed to ensure that the elutant vol does not fall much below 30ul and especially 20ul as this results in poor elution, exacerbating issues with poor recovery from low amounts of starting material To increase the concentration of your total RNA you can precipitate with 3 x volumes of Ethanol @ -80C for 1 hour to overnight 1/10 vol 3M sodium acetate pH 4-5 And CRUCIALLY 1ul of 1mg/ml to 20mg/ml glycogen. The addition of glycogen will greatly enhance your RNA recovery Then, Make cDNA as described sing 1ul of this cDNA mix and then ensure your 3' biased primers to housekeeping genes and GOI, designed to amplify products approximating to 100bp, are exon spanning: This means you specifically target mRNA and not genomic DNA and thus avoid the need to digest with DNASE I to eradicate genomic DNA which can further degrade your RNA and reduce RT efficiency Use 'primer BLAST' in NCBI to make such exon spanning primers: https://www.ncbi.nlm.nih.gov/tools/primer-blast/ These measures should hopefully ensure the Ct of your housekeepers approximates to 20 and that your GOI are 20-30 (ideal range) in concert with a higher concentration of cDNA if you are using a dilution of your cDNA for cardiomyocyte qPCR: I typically use 1ul of the initial 1:5 diluted cDNA reaction mix I should also add Alisha that for cardiomycocytes GAPDH is not your best housekeeper. I deally you should nortmalise for for final analysis with 3 x housekeepers - but that is a whole other subject - and when performing qPCR on HCM, I typically use (and the literature suggests)
Dear Alisha
I have just answered another question about prior cDNA synthesis from cardiomyocyte RNA. I have done this myself in order to analyse gene expression in cardiomyocytes by qPCR
To answer this question:
B Actin
B Tubulin
B2 Micro globulin for example
I hope this helps in conjunction with my previous answer
3 votes
1 thanks
Alisha Fleming
Thank you so much for the detailed answers. I will discuss with my supervisor and hopefully will be able to follow this protocol.
0 votes
0 thanks
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