In fact concentration of RNA for RT ranges from 100 ng to 500ng per reaction. Note that you need to check the quality of RNA. Best way is to check RNA bands in gel. 

Hi

Concentrations (30-50 ng/ul) are enough for real time PCR applications but not enough for RT-PCR applications. In our experience, 200-300 ng/ul enough for getting the desired results in RT-PCR applications. To get high concentrations in RNA extraction, first you can digest your samples with mechanical spalling devices, then you can use RNA extraction kits. We used beaded tubes with lysis buffer, then extract samples with a good commercial RNA exctraction kit, and got high viral RNA concentrations from plant.

Mr.Bavykin and Mr.Şevik,

I used  diluted RNA samples for RT-PCR and checked RNA bands in gel. RNA bands has not enough quality.But I don't apply sequencing DNA... Observing specific area of RNA virus in gel as a PCR produce is  more important for me.In addition, although diluted RNA samples for RT-PCR ,saw specific band of plant virus.

I use  nano drop for measuring RNA con. approximately 260/280 ratio (1.70-2.10) and  RNA amount (15-1000ng/ul).

I use commercial RNA extraction kit and manual method. The first has low RNA concentration,the second has high.

My aim is optimizing RT-PCR and PCR.

Shortly, don't I dilute RNA samples for RT-PCR?

Hi,

My suggestion for optimizing RT-PCR and PCR protocol is using enough RNA or DNA concentration (minimum 200ng/ul), and finding appropriate annealing temperature via gradient PCR. After optimizing, you can find limit of detection (LOD) by testing the diluted samples.

Dear Şevik,

Thank you again your suggestion.I'm going to try to regulate RNA concentration (minimum 200ng/ul). My working primer hasn't enough quality for PCR but I saw a weak band on the gel. İt has G-C ratio %38.It could have been %50-60 but not. Annealing temperature is very important and focus on nevertheless G-C ratio...

In our experience, cDNA quantity would be more important than RNA concentration for qPCR. Until you are loading about 15-20 ng of temple per well, it should be alright unless your gene of interest is of very low copy number. 

Also, if kits give you low quantity and manual methods gives higher quantity for RNA, it is mostly due to contamination during RNA extraction and usually happens especially when RNA concentration is too low.

You can try using Qiagen mini elute columns to get higher concentration and better quality RNA because of low elution volume. Best would be to start with Trizol digestion, and after adding chloroform and spinning them, transfer the upper layer to these mini elute columns and follow their protocols. This way has worked well for us for low yield RNA. 

And inc ase you want to continue with Trizol, you may want to add glycogen along with 100% iso-propanol (after transferring chloroform separated layer in new tube) as it helps in precipitation of RNA and also seeing the pellet after spinning it down. Leave it in -80 degrees over night. Spin the RNA down next day and pellet is easily visible because of glycogen and you can continue with ethanol wash. Just make sure you dry the pellet well after all ethanol washes. 

Good Luck

Dear Mr. Agrawal,

Thank you so much for valuable suggestion. I used chloroform/iso-propanol/ ethanol but it wasn't successful method for me. I'm going on with commercial kit (Thermo Scientific Plant RNA Isolation Kit). Actually it is an appropriate way to ısole total RNA.

I measure total RNA with Nano-Drop,If it indicates 2.10-1.80 (260/280) , it can be successful RT-PCR and PCR. I wonder that  do  I dilute total RNA for good PCR and RT-PCR?

Sincerely

Can you just double confirm that by RT-PCR you mean realtime-qPCR or reverse transcription PCR? 

no no it is not realtime - qPCR ..İt is reverse transcription PCR

Okay. The reason I can think of is the RNA quality in that case. Especially when you don't get bands in few of the samples. Try running the RNA directly on the gel, that would tell you more about the actual concentration and quality. For gel, about 300ng total RNA can be loaded (either in DNA loading buffer or RNA loading buffer) in 1% agarose gel (EtBr or other nucleic acid stain should work) 

Alternatively, some bioanalyzers are available to check RNA quality. 

I have run the RNA on gel, some has good quality but some has not.

I measured RNA concentration using nano drop at 260/280 ration approxmately 1,8-2,10. According to the kit that we used manual, to performe RT-PCR I have to use 30-50 ng/ul of total RNA concentration, do you think this concentration is good enough? All this time, I use pure distilled water to dilute RNA, do you think this way is correct?

for nanodrop it would be essential to look for 230/260 as well, as it denotes contamination. refer to the attached file. 

I think 30-50 ng/microL should be alright for RT-PCR. Which kit do you use?

I use the Thermo Scientific plant rna kit.

One thing to try is to do the RT step at different temperatures.  42-44 C is the suggested temp for our kit.  Use slightly higher temps for RT as it helps eliminate any secondary structure in the RNA.  I think you can try as high as 50 C.  At temps this high or higher you risk denaturing the RT.  Also, be sure and DNase treat the RNA with RNase-Free DNase.  When I use kits I often do an extra wash step at the "wash"  step in the protocol.  Hope this helps.

Dear Mr. Wilkinson,

First of all , thanks your suggestions....I use 42C for RT-PCR step and RNase Free water...

I try to optimize RT-PCR and PCR everyday...I think that  problem is RNA concentration  in RT-PCR tube and its concentration is final volume in the mix 30-50ng/ul. So my total RNA concentration 300-1000 ng/ul in stock tube .

I use 1 or 2 ul from Total 300-1000 ng/ul RNA for 25ul RT -PCR mix...İs it correct?

Dear Hall,

Thanks your valued suggestion.

I should take account of the suggestion. I know that It is important 260/280 ratio but not 260/230...So do I take total(stock) RNA 1-2 ul for RT-PCR, don't I? İt is only important that for pure RNA 260/280 and 260/230, isn't it? Also do I dilute high conc. RNA (600-1500 ng/ul) for RT-PCR?

Dear Mr. Hall ,

Thank you again...I'm getting understand now. I did RNA isoaltion yesterday and saw low conc. RNA but high 260/280 and 260/230.For example; 177 ng/ul RNA conc. 260/280 -2.10 and  260/230 >1 .I will try to do RT-PCR again again...İf I see good results the PCR it is ok for me...

Dear Mr. Hall,

Thank you again..You are right for those ratios...

I will try to make optimisation for RT-PCR and PCR...I haven't just see the specific bands on gel yet.

RT-PCR made with 200-300 ng/ul RNA in total volume will yield accurate results.

Hi Soliman

I saw lots of values about RNA con. I think , RNA 260/280 and 260/230 are very important for PCR...

They must be clear values...For example; 260/280 1,98  and 260/230 1,80 ...

Thank you your answer...

Hello Mr. Hall,

Thank you your advices...

Moreover, I use your suggestions in my studies (RNA extractions) ...İt is important for me...I learned RNA values thanks to you...