Detection of titer and purity of recombinant Adeno-Associated Virus

     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
1	Marker
2	1-196-T181110	rAAV-CAG-DIO-Gcamp6s-WPRE-pA
3	Positive control 157	rAAV-nEf1α-FDIO-TVA-P2A-NLS-dTomato-WPRE-pA

(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. 

2. 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. 

3. 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 

 

2. Standard curve 

 
 

3. 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

 
 

4. 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) 

2.  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.