RNA isolated from cell line and got a concentration of about ~ 300 ng / ul (purity of about 2.0. The RT-PCR must have a mix of 10 ul + 10 ul RNA wheel RNA - 1 ug.
I performed the operation 1000ng / ul to 300ng. 3.3 ul RNA complete + 6.7 ul water.
I getting cDNA. And now I have a question?
I give 1/2 ul cDNA to qPCR - if I have to measure its concentration, as ssDNA? Can I immediately take the 1ul cDNA for qPCR ithout measuring?
I measured the test and got a cDNA having a concentration of 1510 ng / ul and I do not know if I have a diluted sample, eg. To 100 ng?
Thank for help
The amount of RNA you have used in cDNA synthesis is the total RNA content of the cell e.g. rRNA, tRNA, mRNA etc. Now, considering that you are using oligo dT primer for cDNA synthesis, the cDNA hence formed will be from mRNA.
For qPCR quantification of cDNA if you are using relative quantification method the amount of template you are using does not required to be same(means no need to measure) because you have to quantified in reference to internal control like beta actin, GAPDH etc. For your convenient you can use equal amount of cDNA for each reaction.
Wish you all the best.
The amount of RNA you have used in cDNA synthesis is the total RNA content of the cell e.g. rRNA, tRNA, mRNA etc. Now, considering that you are using oligo dT primer for cDNA synthesis, the cDNA hence formed will be from mRNA.
For qPCR quantification of cDNA if you are using relative quantification method the amount of template you are using does not required to be same(means no need to measure) because you have to quantified in reference to internal control like beta actin, GAPDH etc. For your convenient you can use equal amount of cDNA for each reaction.
Wish you all the best.
As stated above... when using relative quantification measuring cDNA is not always necessary.
If you always use the same amount of RNA for RT and process the same... than you can use a fixed amount (volume).
More important is to realize that when performing qPCR on different samples, you also have to be sure about your RT-reaction consistency. So, perform RT on your samples all at the same time with the same mix, or re-run an old sample again to verify.
Also, when performing qPCR I would advise on not pipetting too small volumes. Pipeting 0.5 µL is asking for variation in your replicates! You can your cDNA, so in each reaction you can then pipet a larger volume to get better consistency (I use 2 µL as minimum!).
Also, when not dilluting you might get inhibitory effects of your RT-components in the qPCR reaction.
Dear Marcin
- No you do not need to measure the concentration of cDNA; Just your starting RNA by Nanodrop:
- The custom is to use a set amount of total RNA - usually 500ug to 2ug (for low copy number genes) - and then a set amount of un purified cDNA: Usually 1ul per qPCR reaction
- In fact, if you measure your cDNA you require fluorimetry - e.g. the Qubit sybr dye system - otherwise the bulk of your reading will be constituted by unincorporated dNTPs (in the case of 260nM reading by Nanodrop)
- Thus you can measure your cDNA by fluorimetry and then adjust the amount of cDNA to a set amount. The advantage of this is that it takes into account differences in cDNA efficiency synthesis linked to the purity of your starting RNA template
- Alternatively, if you purify your cDNA this will remove unincorporated dNTPs permitting standard Nanodrop readings at 260nM as you would procure for extracted RNA, e.g
- https://www.thermofisher.com/order/catalog/product/AM1756
- Alternatively, there are many one step RT PCR kits that end with column purification of derived cDNA. This there again will allow you measure the concentration of your cDNA; the MACS kit from miltenyi Biotech is one I used a lot in a previous position:
- http://www.miltenyibiotec.com/en/products-and-services/macsmolecular/nucleic-acid-research/mrna-research/macs-and-multimacs-mrna-isolation-kits.aspx
- However, most labs simply perform RT from a set amount of total RNA and then add 1ul of cDNA to resulting qPCR reactions
- Regarding differences in cDNA synthesis efficiency these are actually accounted for during normalisation with housekeeper genes so that becomes a redundant issue
- Badges
- Science topic