The M-MLV system is a reverse transcriptase enzyme used for the synthesis of complementary DNA (cDNA) from RNA templates. The M-MLV enzyme catalyzes the conversion of single-stranded RNA into double-stranded cDNA, which can then be used for various downstream applications, such as gene expression analysis, cloning, and sequencing.
The RNA were extracted from the three viral stocks (AnD133719, SHM172805 and ArD141967) and were used as templates for RT-PCR. Specific primers (Table 2), the M-MLV system (Invitrogen, Carlsbad, CA, USA), and the Go-Taq PCR Kit (Promega, Madison, WI, USA) were used for cDNA synthesis and amplification, according to the manufacturer’s instructions. All primers used to amplify the S and M segments were designed according to RVFV sequences available in GenBank. Accession numbers of all RVFV sequences used to design the primers are presented in Additional file 1: Table S1. The PCR products of the expected sizes were purified directly from the agarose gel using a QIAgen Gel extraction kit and sequenced by Cogenics (Beckman Coulter Genomics, Essex, United Kingdom). Sequencing was performed in both directions, using the original reverse and forward primers as for the amplification.
List of primers used
Nom
Segment
Position
Sequence
Tm
NSng
S
31–48
TATCATGGATTACTTTCC
48
NSca
S
841–824
CCTTAACCTCTAATCAAC
50
M1F
M
3–22
ACAAAGACCGGTGCAACTTC
53.9
M1R
M
1120–1140
CCAYGCAAAGGGTATGCAAT
53.2
M2F
M
1035–1054
TGAGGACTCTGAATTRCACCT
48.7
M2R
M
2395–2415
TCCAGAGAGTTGAGCCTTGC
53.3
MRV1a
M
3050–3068
CAAATGACTACCAGTCAGC
44.6
MRV2g
M
2262–2292
GGTGGAAGGACTCTGCGA
52.5
M3F
M
2979–2998
CAGTCCTCAGTGAGCYCATA
46.1
M3R
M
3763–3782
TCTCGGTTCTGGRGTGTGAA
52.5
Ndiaye E.H., Fall G., Gaye A., Bob N.S., Talla C., Diagne C.T., Diallo D., BA Y., Dia I., Kohl A., Sall A.A, & Diallo M. (2016). Vector competence of Aedes vexans (Meigen), Culex poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African lineages of Rift Valley fever virus. Parasites & Vectors, 9, 94.
RNA of cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Concentration and purity were measured by spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Totally, 500 ng RNA was reverse transcribed into cDNA by M-MLV system (Cat no. C28025-011; Invitrogen, China). PCR reactions were performed by the 7900HT Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR-green (TAKARA, Japan) in a 10 μl reaction mixture. The primers are shown in additional information (Supplementary Table 2).
Wei L., Ma X., Hou Y., Zhao T., Sun R., Qiu C., Liu Y., Qiu Z., Liu Z, & Jiang J. (2023). Verteporfin reverses progestin resistance through YAP/TAZ-PI3K-Akt pathway in endometrial carcinoma. Cell Death Discovery, 9, 30.
Total RNA was purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using the M‐MLV system (Cat no. C28025‐011; Invitrogen, China). qRT‐PCR quantified RNA expression using the SYBR Green Master Mix (Takara, Japan). ACTB and U6 were used as internal controls for mRNAs and miRNAs, respectively. The relative RNA abundance was calculated using the standard 2‐ΔΔCt method. The primers used in the present study are listed in Table S2. Plasmid extraction was performed according to the manufacturer’s protocols (Omega Bio‐tek, Norcross, GA, USA).
Kong D., Hou Y., Li W., Ma X, & Jiang J. (2021). LncRNA‐ZXF1 stabilizes P21 expression in endometrioid endometrial carcinoma by inhibiting ubiquitination‐mediated degradation and regulating the miR‐378a‐3p/PCDHA3 axis. Molecular Oncology, 16(3), 813-829.
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