I have produced lentivirus with my gene of interest. the protocol refers to some papers(Kutner, R., Zhang, X.-Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nature Protocols 4, 495–505 (2009).) My question is how to perform the qPCR and analyze the result?(I can not understand the formula of this paper)
I purchased qPCR assay of GAG and RNaseP
Review of the previous experiments:
Day0: Seed 5*104 U87 cells/well in each 6 well plate.
Day1: Harvest 10ml supernatant containing LV from 293T cell culture. And concentrated to 100ul, split to 20ul/tube and store in-80’C. Dilute this stock 1ul in 99ul PBS, and add 0.5ul, 5ul, 50ul to designed U87 cell in 6well plate (each well has 1.2*105cell). One well is no infection ctr.(only add Polybrene).
Day2: Replace U87 cell medium to remove LV.
Day5: Harvest U87(96 after infection), and extract gDNA, then store in -20’C.
qPCR reaction
*qPCR assay of GAG and RNaseP(purchased from Life)
Result of qPCR
Mean Cq(GAG) Mean Cq(RNaseP)
0.5ul 29.71 23.95
5ul 26.95 24.01
50ul 23.97 24.11
* this is the Data analysis of the protocol:
"vector copy numbers in HOS cells are normalized to human RNaseP gene copies and presented as proviral copies per genome equivalent. Calculate titers (integration units per ml, IU/ml) according to the following formula:
IU/ml= C x N x D x 1,000)/V, where C = proviral copies per genome, N = number of cells at time of transduction (corresponding to about 1 x 105 HOS cells per well), D = dilution of vector preparation, V = volume of diluted vector
added in each well for transduction. "
summarize of my question:
1. What is the meaning of the 1st sentence? is that mean C value=2^(CqRNaseP-CqGAG) ???
2. This protocol dosen't need standard curve(I think). is the method which use standard curve is more accurate? now I have my plasmid of lentivector which contain GAG, so do I need to purchase a plasmid containing RNaseP?
3. Briefly speaking, how to analyze the qPCR result for LV titration?
Looking forward your help! thanks!
Dear Linchun,
Here is my protocol of lentivirus titration with fluorescent microscopy.
a. Seed 293T 1×10^4 cells/well in a 96-well plate one day in advance.
b. Perform gradient dilution of the lentiviral particles to 1:10, 1:100, 1:1000, 1:10^4, 1:10^5, 1:10^6 in 100ul final volume in culture medium.
c. Total 100μl viral particle mixture should be added to each well with at least 3 replicates per virus.
d. Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%-30% positive cells/well). Count the triplicates and average the number of positive cells.
e. Estimate the lentivirus titer using the following formulation: Viral titer (TU/ml) = number of fluorescent positive cells × 10 × dilution.
Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
HI, you can perform qPCR for WPRE.
Check this paper for primers and probes
http://www.ncbi.nlm.nih.gov/pubmed/23881415
we usually compare titers to a reference batch of LV-GFP that has been tittered by FACS and all our tigers are relative to the reference batch.
ATB,
Luis
Hello,
We use the protocole described in this paper
http://www.nature.com/nprot/journal/v4/n4/full/nprot.2009.22.html
With a primer between the LTR and PSI regions that the sequence is in this paper
http://www.biomedcentral.com/1472-6750/6/34
(If you need access to PDFv tell me)
Like Luis we compare titers to a reference GFP vector. The best way is to produce the reference(s) vector(s) in the same time that the others
Hope this help you!
Best,
Nicolas
Hi, Luis,
Thank you for your help! I looked into your paper of The American Society of Gene & Cell Therapy.
I have a question, in the part of "Materials and Methods-Cloning and lentiviral vector production". what's the meaning by " Te resulting values were then used to calculate the estimated titer of each lentiviral vector relative to the pHR-CMV-GFP titer."?
I use GAG gene as the target of integrated LV and RNaseP as the internal control, just similar as your WPRE and Albumine.The qPCR method is really similar.
Can you tell me more detail about how to do the qPCR analysis for titration? like formula?
I am a really freshman in LV field, and these questions will help for guys like me.Thanks a lot!
check this excel sheet:) hope it helps as it has all the calculations used.
ATB,
Luis
Sorry, I still can not figure out what is the C value in the fomula as described"vector copy numbers in HOS cells are normalized to human RNaseP gene copies and presented as proviral copies per genome equivalent"
Who can answer that?
I think they divide ct values of VC/RNase P gene, similar to what we do with wore/albumin. That would be my interpretation.
Hi, Luis
In your Excel, what's the meaning of value of "delta delta CT" & "REF TITER"?
I know, your delta ct is "Av CTwpre- Av CTalb", and delta delta CT is "Av CTwpre- Av CTalb" -- (2.01), where is this 2.01 from?
And is that "REF TITER" is from your titer of GFP Reference Lentivirus?
Thank you!
Hi,
as the specific conditions of each cPCR reaction vary, we need to set one reaction as a normaliser. in our case the highest concentration of REF LV. In that reaction the dCT was 2.01. We then use this value to calculate the ddCT across the qPCR run.
Ref Titer is the tiger of the reference batch
Hope it helps,
Luis
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