Total RNA was extracted from Pmel-1 cells 24h after electroporation with the Maxwell RSC simplyRNA extraction kit (Promega), according to the manufacturer’s instructions and subsequently retrotranscribed into cDNA using M-MLV enzyme kit (Invitrogen). Real-time PCR reactions were performed into Bio-rad CFX qPCR system with customized primers for mouse Mir155hg (FW 5′-AAACCAGGAAGGGGAAGTGTG and Rv 5′-TAGGAGTCAGAGGCCAA), mouse Inpp5d (FW 5′-TCCCCAGATCAGCAACTCAC and Rv 5′-CAGATCCCCAGGTCTTGCCT) and mouse Actb (FW 5′-CGCGTCCACCCGCGAG and Rv 5′-CCTGCCTAGGGCG).
M mlv enzyme kit
Manufactured by Thermo Fisher Scientific
The M-MLV enzyme kit is a reagent used in molecular biology laboratories for the reverse transcription of RNA to cDNA. The kit contains the M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase enzyme, which catalyzes the conversion of single-stranded RNA into double-stranded cDNA, a necessary step for various downstream applications such as gene expression analysis and cDNA library construction.
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Lab products found in correlation
Cfx qpcr system, by Bio-Rad (1 mentions)
Maxwell rsc simplyrna extraction kit, by Promega (1 mentions)
Lightcycler 1, by Roche (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
2 protocols using m mlv enzyme kit
1
Analyzing miRNA Expression in Pmel-1 Cells
2
Quantitative Real-Time PCR and Immunofluorescence Analysis
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Real-time qRT-PCR Total RNA was isolated using Trizol (Invitrogen), treated with TURBO DNase (Ambion) and 500 ng was reverse transcribed using an mMLV enzyme kit (Invitrogen). PCR amplification of cDNA was performed using a synthetic external and nonhomologous poly(A) standard RNA (smRNA) to calibrate the efficiency of the reverse-transcriptase (RT) step (Morales et al. 2006) (link). Quantitative amplification of target cDNAs was determined using a QuantiTect SYBR Green PCR kit (Qiagen) on the Lightcycler 1.2 (Roche Diagnostics) as described (Marcel et al. 2013) . Primers are listed in Supplementary Tables 2 and3.
Immunofluorescence Cells grown on glass coverslips were fixed with paraformaldehyde (PFA 4%, pH 7.4), permeabilized (Triton X100, 0.5%) and blocked (BSA, 1%). β-catenin (1:300) and WNT4 (1:100) primary antibodies were from Santa Cruz Biotechnology. Alexa488 (Invitrogen), Alexa633 (Interchim) and 4-6-diamidino-2phenylindole (Vector Laboratories, Burlingame, CA, USA) were visualized by confocal microscopy (Carl Zeiss 510).
Immunofluorescence Cells grown on glass coverslips were fixed with paraformaldehyde (PFA 4%, pH 7.4), permeabilized (Triton X100, 0.5%) and blocked (BSA, 1%). β-catenin (1:300) and WNT4 (1:100) primary antibodies were from Santa Cruz Biotechnology. Alexa488 (Invitrogen), Alexa633 (Interchim) and 4-6-diamidino-2phenylindole (Vector Laboratories, Burlingame, CA, USA) were visualized by confocal microscopy (Carl Zeiss 510).
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