My cDNA synthesis kit said that the maximum amount of RNA should be used in cDNA synthesis is 5μg, so I want to know if I use more than this amount and do Real-Time PCR after that, will it cause any problem in Real-Time PCR result? Cause I think it may increase the CT result in qPCR.
Thank you
Hi Ali,
recommandations are given to optimize your preparations, but if you increase the quantity of RNA to produce the cDNA, make sure to do it strictly in all samples to avoid bias introduction (failures of the production or sur-representation of one cDNA to the other).
fred
Hi Ali,
recommandations are given to optimize your preparations, but if you increase the quantity of RNA to produce the cDNA, make sure to do it strictly in all samples to avoid bias introduction (failures of the production or sur-representation of one cDNA to the other).
fred
Dewar Ali
- For high copy number genes and modern RT kits you only need up to 1ug of total RNA
- For low copy number genes up to 2ug
- There is abolsutely nothing to be gained from using > 2ug; It will not increase your sensitivity and just waist your RNA
- If you qPCR doesnt work with 2ug of total RNA then the problem is related to something else like RNA quality; primer efficacy and not a puacity of starting material:
- Make sure your RNA has a 260/280 > 1.8 and a 260/230 > 1.0. In particualr if the latter is < 1.0 and especially less than 0.5 you have slat contamination in your RNA that will result in poor cDNA production even withn 5ug of RNA; Indeed using more RNA will excarebate the effect of salt with such a prep
- In this situation your only option is to make fresh RNA and make sure you include 2 x ethanol based wash steps in your extraction procedure and if needs be take your purified RNA and either put down a second column or precipitate with ethanol/isopropanol and wash your pellet x 2 with ROOM TEMP 70 % ethanol
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