Help with quantitative RT-PCR and RNA standards

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dga2000
dga2000's picture
Help with quantitative RT-PCR and RNA standards

I am beginner lab technician with qRT-PCR and I sick help.  I  would appreciate if you can advice me on the way I proceeded with my first assay. I am using a FAM-TAMRA TaqMan probe in a Chromo4. In the plate protocol set up, I specified FAM as the only fluorophore, and did not include TAMRA.
I extracted RNA from 140 uL a virus preparation that was previously titrated as 10^4 PFU/uL.
After extraction, I eluted RNA in 60 uL elution buffer and measured concentration (70ng/uL) to use it for the standard curve.
I am using 5uL of RNA in a 50 uL final volume of One-Step qRT-PCR reaction.
I set up the standard curve as PFU. For this, I multiplied 10^4 PFU/uLx140uL and divided by 60uL, then multiplied by 5uL to get the total PFU per reaction.
I made 6-fold dilutions of the undiluted RNA to prepare the quantification standards (Qs) corresponding to 116667, 11666.7, 1166.67, 116.667, 11.6667, 1.16667 PFU.
I used in the first try, the Qs, the NTC and the undiluted RNA as positive control.
The equation that I got was y=-0.3012+12.93 with r^2=0.872
Average values of Ct were 22.52 (SD 0.91), 25.90 (SD0.85), 29.30 (SD 1.86), 30.58 (2.85), 33.56 (2.12), 37.32 (1.69) for Qs from undiluted on, and 19.40 (SD 2.52) for the positive control. SD: Standard deviation. 
The average PFU for QS were 116667, 11666.7, 1166.67, 116.667, 11.6667 and 1.16667, respectively, and 2.084e+007 for the positive control.
In the standard curve graph, all the Qs were out of the linear regression. Is there any advice to improve it, please?
To be able to calculate the copy number, should I cloned the cDNA? Any advice on this matter?
Thanks you for your help,
Digna