Hello, I have two questions here. 1st is it important to equalize RNA concentration for cDNA synthesis in case of gene expression? I do calculation using formula Vol1 *conc1= Vol2 *conc2 , but i always get low concentration that i want, why what is reason?
2nd question is, is any one use QIAGEN RNase free DNase set to remove genomic DNA, Is it good in case of RNA yield & degradation after removing genomic DNA? Which step is critical during using this kit?
Hi,
For your study of gene expression you do not need to equalize the amount of RNA because your are using some reference genes in you Q-PCR. For the second question I suggest to do exactly what the vendor recommend and I thing QIAGEN recommendations are good.
Good a luck
Hi,
I just want to make sure I understand. By saying equalizing the RNA concentration for cDNA synthesis, do you mean for example, that you are using 1ug for all your runs?
If so, are you using this formula: e.g. 20ng/ul x volume= 1ug/ul, with this you must remember to convert from ng/ul to ug/ul (do so by /1000).
Also, if your RNA yield was very good, e.g. 1000ng/ul, you may want to dilute your RNA first before using it for cDNA synthesis, as this will just make it more accurate (difficult to pipette small volumes).
For the second question, I agree with Abderrahmane, QIAGEN recommendations are good. I used the RNeasy Plus from Qiagen and also tested gene expression, and it worked well. I don't know much about the QIAGEN RNase free DNase though.
Hope this helped!
Good luck!
I mean is it important that i bring all samples RNA concentration same e.g all samples concen become 500ng/ul, OR i take different RNA concentration but for cDNA synthesis take same amount e.g 1 ul?
I usually used 1ug of RNA for cDNA synthesis, and equal amounts of my cDNA also.
Hello Bushra,
I usually use Takara to treat my RNA samples with DNase, the equalisation story at this step goes back to the fact that DNase (like any other enzyme which you use), has a efficiency level which is indicated in the instructions of your enzyme, for instance, in case of DNase which i use, i can use 30-50ug of RNA to be treated with 1 unit of DNase (it means if i have more RNA, they do not guarantee if all the DNA is degraded after the treatment with this enzyme). So it's always better to check the OD of your samples and equalise them as per the instructions of the DNase you're using.
the next step would be reviving your RNA after the DNase treatment and cDNA synthesis. It DEFINITELY is important for you to equalise the OD of your samples before the cDNA synthesis as it is the main point of qRT-PCR. you later on want to compare the expression level of your genes or whatever you're working on based on their real expression level, so that step is a MUST.
If your RNA yield is low, you may try using more cells/bigger tissue for RNA isolation or check which other step you can do better in order to improve your final OD.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology