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Detection of titer and purity of recombinant Adeno-Associated Virus

Published On 09/23/2019 12:47 AM

Detection of titer and purity of recombinant Adeno-Associated Virus

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 1. Detection of rAAV titer by SYBR Green qPCR
     In general, rAAV of various serotypes contain either the ITR sequence (partial sequence of the adeno-associated virus genome) or the WPRE (viral titer enhancer element) of serotype 2. We used the SYBR Green qPCR method. In order to quantify the copy number of recombinant adeno-associated virus genome and determine the titer of recombinant adeno-associated virus, a pair of primers specific to the ITR/WPRE sequence of recombinant adeno-associated virus type 2 were designed to detect the copy number of ITR/WPRE sequence.
Date: November 15, 2018        Product batch: 1-196-T181110  operator: Yangxiaohao 
Product Name 1-196-T181110 
rAAV-CAG-DIO-Gcamp6s-WPRE-pA 
Product batch  1-196-T181110 
Titre (vg/ml)    5.56E+12
Packing specification (ul) 20 
 
 2. Identification of the purity of recombinant adeno-associated virus by SDS-PEGA and silver staining. 
Product batch: 1-196-T181110 
Lane  Sample  Full Name of Sample 
Marker 
1-196-T181110 rAAV-CAG-DIO-Gcamp6s-WPRE-pA
Positive control 157  rAAV-nEf1α-FDIO-TVA-P2A-NLS-dTomato-WPRE-pA
 Identification of the purity of recombinant adeno-associated virus by SDS-PEGA and silver staining(0 times diluted) 
(Note: As there are samples of other customers on the same film, this picture is a spliced picture. If you need the original picture, please contact Brain VTA technical support ) 
Conclusion: 1. The size of the three protein bands in the lane of the sample showed that the adeno-associated virus coat VP1,VP2,VP3 was the same as that of the adeno-associated virus coat, and the content ratio was about 1 / 1 / 10, which was similar to the composition of the adeno-associated disease albumin coat. 
2. There were no other distinct protein bands in the sample s lanes except for the three distinct protein bands of VP1,VP2,VP3. 
Method of experiment: 
1.  A quantitative  
Sample (1196T181110, positive 2ul/ pore 
Standard sample (1.0E+08 copies/2ul,2ul/ pore
 Protocol: 
  1. sample preparation
The 2ul samples were diluted to 20ul, mixed with 5ul nuclease 0.2ul, 37 , 1h, then mixed with 5ul 2m NaOH, in 55  water bath for 30min, centrifuged. 5ul 2m HCl was added to the centrifuge tube to mix well. Add 135ul ddHO, to mix, the sample is the sample to be tested. 
  1. preparation of standard (STD): 
The standard solution of 10ul was diluted to 2.0E+08copies/uls by adding 90ul ddH2 According to this method, five times of dilution were repeated, and a series of concentration gradients were obtained, including 2.0E+08copies/ul2.0E+07copies/ul2.0E+06copies/ul2.0E+05copies/ul2.0E+04copies/ul2.0E+03copies/ul. 
  1. SYBY Green qPCR 
Q-PCR reaction using primers specifically binding to the WPRE gene region of adeno-associated virus. 
Forward primer WPRE: 5-CCGTTGTCAGGCAACGTG-3' 
Reverse primer WPRE: 5-AGCTGACAGGTGGTGGCAAT-3'
Reagent  Vol. per reaction 
SYBR Green PCR Mix (2X)  5ul 
FWD WPRE (20uM)  0.2ul 
REV WPRE (20uM) 0.2ul 
Nuclease-free water  2.6ul 
Sample DNA  2ul 
 
 
 
 
 
 
 
SYBR Green q-PCR reaction conditions 
Pre-denature:    95°C 3 min                 40 cycles 
                                     95°C 15 sec                 60°C 1 min
 
  1. Sample amplification curve 
 
Sample amplification curve
  1. Standard curve 
 
 
  1. q-PCR data
 
Well  Fluor  Target  Content  Sample  Cq  Cq Mean  SQ Mean
A01  SYBR  WPRE  Std-1   
 
 
11.23   11.33  1.00E+08 
A02
 
 
SYBR
 
 
WPRE
 
 
Std-1
 
 
 
 
 
11.43
 
 
11.33
 
 
1.00E+08
 
 
B01
 
 
SYBR
 
 
WPRE
 
 
Std-2
 
 
 
 
 
15.61
 
 
15.52
 
 
1.00E+07
 
 
B02
 
 
SYBR
 
 
WPRE
 
 
Std-2
 
 
 
 
 
15.42
 
 
15.52
 
 
1.00E+07
 
 
C02
 
 
SYBR
 
 
WPRE
 
 
Std-3
 
 
 
 
 
19.16
 
 
19.18
 
 
1.00E+06
 
 
C01
 
 
SYBR
 
 
WPRE
 
 
Std-3
 
 
 
 
 
19.20
 
 
19.18
 
 
1.00E+06
 
 
D02 
 
SYBR 
 
WPRE 
 
Std-4 
 
 
 
 
22.96 
 
23.08 
 
1.00E+05
 
D01
 
 
SYBR
 
 
WPRE
 
 
Std-4
 
 
 
 
 
23.20
 
 
23.08
 
 
1.00E+05
 
 
E02
 
 
SYBR
 
 
WPRE
 
 
Std-5
 
 
 
 
 
26.24
 
 
26.40
 
 
1.00E+04
 
 
E01
 
 
SYBR
 
 
WPRE
 
 
Std-5
 
 
 
 
 
26.57
 
 
26.40
 
 
1.00E+04
 
 
F02
 
 
SYBR
 
 
WPRE
 
 
Std-6
 
 
 
 
 
31.04
 
 
31.05
 
 
1.00E+03
 
 
F01
 
 
SYBR
 
 
WPRE
 
 
Std-6
 
 
 
 
 
31.06
 
 
31.05
 
 
1.00E+03
 
 
C05
 
 
SYBR
 
 
WPRE
 
 
Unkn-3
 
 
1-196
 
 
14.89
 
 
14.75
 
 
1.40E+07
 
 
C06
 
 
SYBR
 
 
WPRE
 
 
Unkn-3
 
 
1-196
 
 
14.61
 
 
14.75
 
 
1.40E+07
 
 
 
 
  1. Titer calculation 
Sample Number Average CQ value SQ Mean(vg/ul) 
 
Dilution multiple
 
 
q-PCR Titer
 
 
(vg/ml)
 
 
Calibrated titer(vg/ml)
 
 
Post-loading titer(vg/ml)
 
 
PT-0196
 
 
14.75 
 
 1.40E+07                                                                                                                                  
 
 
900 
 
1.26E+13 
 
8.34E+12 
 
5.56E+12
 
 
(the sample is 1.5 times diluted) 
  1.  Identification of adeno-associated virus purity by SDS-PEGA 
    1. SDS-PEGA electrophoresis, AAV sample 10ul/ pore, 1E11vg, protein marker1ul/ pore, 120V electrophoresis 1h. 
    2. decolorizing after silver staining and taking pictures. 
This entry was posted in Manuals and Protocols

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