Also, the overwhelming opinion has been - dont measure the cDNA. Measure the RNA, load a known amount into your RT reaction and then use a set volume in your PCR. Will be more accurate. Loading a known volume into the RT means you wont overload the enzymes leading to a poor reaction and/or keeping your samples within the optimal mid range of your standard curves during the PCR.

single-stranded DNA.

You should go for ssDNA. Does this doubt come in your mind because of imagining DNA-RNA hybrid?

@Neelofer Mirza: while estimating cDNA with nanodrop, there is interference of left over dNTPs fluctuating the values and I dont think that one can get a reliable value. Instead if you are preparing cDNA from calculated values of RNA there is no need of estimation of cDNA.

if you confused with DNA-RNA hybrid, break the hybrid at 95'c(2 min) as ur final step of cDNA synthesis

i agree with Muhammad S Masoud. you have to calculate only your RNA concentration.

Thanks all. @Swapnil-yes,we got confused due to DNA-RNA hybrid. @Dhanasekaran- dats a gud idea.

@Muhammad S Masoud- thanks, I also thought so.

Can u people help me further,

I took 2000 ng of RNA for preparing each cDNA . Now I hv to go for real time PCR. Since, d amount of starting material should b same i.e. d DNA template, so we thought we might need to quantify d cDNA. But I really got confused when I saw d difference in quantification, most of the samples hv readin b/w 1500-2000 ng but few had above 2000ng and still a few had readings in d range of 400-500 ng. How should I proceed for real time PCR?? :(

if you still have doubt why not amplify your house keeping gene (GAPDH OR beta actin) and analyze on the gel that either they have the same band intensity

well, I just did it, I amplified Tubulin gene, most of d samples r givin similar intensity bt few r not. should I proceed?

I think you should proceed because when you will run the realtime, it will automatically normalized the values

Ok, thanks.

@Muhammad S Masoud: You can't observe this difference on gel with end product of conventional PCR as it is in plateau phase. That is the one of the main reason we prefer qPCR. By my logic, as the left over quantity of dNTP will be present in all samples, nanodrop can give closer to actual RELATIVE concentrations of all cDNAs. OR if you want exact concentration of cDNA one can try cDNA purification protocol before taking nanodrop reading. @Neelofar Mirza: When I faced the same problem I diluted the RNA so as to achieve near to equal concentrations of all RNAs. @Muhammad S Masoud: I couldn't understand how can the values be normalized? If the concentrations of samples are varying in such broad range, CT value will be definitely affected. Isn’t it?

@Kadam: yes CT values definitely affected but I meant the normalization of gene expression against house keeping gene (control)

@Swapnil- as I hv mentioned, I quantified all my RNA samples and hv taken 2000ng of each sample for cDNA prep.

Can u people send me sum gud papers so that I can understand qPCR well before actually doing it.

@Muhammad : Exactly! Normalization WITHIN the one RNA sample! But with variable RNA concentrations you can't check the expression ACROSS the samples!

@Neelofar : As you have taken the 2000ng RNA of each RNA sample you dont have to worry. As we were discussing about cDNA quantification, if you want to quntify cDNA, do cDNA purification and then take nanodrop reading. Here is a link for online tutorial about qPCR-http://pathmicro.med.sc.edu/pcr/realtime-home.htm >For online discussion forum where some basic questions are already asked and answered- http://www.genetargetsolutions.com/pages/Real%252dTime-PCR-FAQS-.html >One of simple paper available on net (There are many useful references in this article)- Basic principles of real-time quantitaive PCR. Manit Arya, Iqbal s Shergill, Mangali Williamson, Lyndon Gommersall, Neehar Arya and Hitendra RH Patel I hope you will find it useful.

@Swapnil- thank u so much!! Wat kit would u suggest for cDNA purification??

Hi, QIAquick PCR Purification Kit (QIAGEN) gives good results. There is one patented technology *Magtration® -cDNA Purification (12XP). You can find the details about this patent with- Japanese Patent: No.3115501; US Patent: No. 5,702,950; European Patent No.687501.

Nanodrop gives fake result of quantification of cDNA . So its better to take Total RNA concentration from nanodrop . mRNA is about 10% in case of eukaro and about 5% in proka of total RNA.

Thanks.

I don t understand why one can t compare samples that derive from RNAs of different concentrations. Normalization of gene expression is meant to correct such discrepancies, e.g. if sample A has higher RNA concentration of sample B, then the reference gene in sample A will be more abundant than in sample B and therefore will correct the difference. Am I wrong??

Of the 19 answers and contributions to the question by Neelofer Mirza, none actually has said whether he should read cDNA as ssDNA or dsDNA. I am equally interested.

cDNA is single stranded, therefore use ssDNA. Cheers.

Also, the overwhelming opinion has been - dont measure the cDNA. Measure the RNA, load a known amount into your RT reaction and then use a set volume in your PCR. Will be more accurate. Loading a known volume into the RT means you wont overload the enzymes leading to a poor reaction and/or keeping your samples within the optimal mid range of your standard curves during the PCR.

Hi, i have a question here. For the RT-PCR, it is almost impossible guarantee the efficiency of run is 100%, right? If let say we standardize the amount of RNA loaded for RT-PCR, will it be like getting variation in amount of cDNA? Will it resulted various in amount of cDNA loaded for qPCR as well?

Thanks.

you cannot spec 260/280 from first strand synthesis of cDNA, RNA and unincorporated dNTPs will also give an absorbance, if you really want to quantify it then a fluorescent assay such as oligreen from life technologies is the way forward, i myself hold with the recommended method of normalising mRNA going into the RT reaction then use a house keeping gene to demonstrate the consistency of the rt reaction, Densitometry can be used to normalise these data and any other experimental data sets collected by pcr.

@Tzu Shan Ng

Yes, there will be variation in your cDNA amounts inevitably. However, remember that you also normalize your GOI expression to your reference gene; therefore if you have more cDNA in one reaction because of higher reaction efficiency, the GOI expression will be normalized to the higher reference gene expression and thus the results will be comparable.

Is to possible to measure amount of cDNA or atleast equalize different samples using house keeping genes lik eGAPDH or RnasP

.........................

by using absolute quantification on real time PCR

hello, today at the lab meeting I have been told that iNTO should not interfere much with a reading of the cDna at a nanodrop.

Also, in my experience it is important to get all the gapdh values of the q-for in between no more than 1CT of difference. Normalizing the results of a q-pcr to very fluctuating gapdh values can get to false results. 

Into was supposed to be dNTP 

"hello, today at the lab meeting I have been told that iNTO should not interfere much with a reading of the cDna at a nanodrop."

Whoever told you this is wrong, dNTPs will absorb more than a ds polymer of the same concentration due to base stacking and the hyperchromic effect... undergrad level biophysics ... 

http://en.wikipedia.org/wiki/Hyperchromicity