Journal: PLoS ONE

Article Title: Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

doi: 10.1371/journal.pone.0022822

Figure Lengend Snippet: Total RNA was extracted from MA-10 cells and treated with DNase I. 5′ RACE was performed using three different sets of three sequence-specific primers for RT, PCR, and nested PCR. Following agarose gel electrophoresis, bands were eluted, and cloned into the pGEM®-T Easy vector for sequencing. The results were analyzed by BLAST and Vector NTITM Suite 8 software. A . Upper panel. Schematic diagram showing the relative location of the three groups of primers (G1, G2, and G3) used. Lower panels. Representative images of the nested PCR products generated by amplification with each primer group (lane 2). Lane 1 shows the DNA molecular weight ladder. Arrows indicate the apparent sizes (in base pairs, or bp) of the eluted bands. B . Schematic diagram showing the representative sequences of the resultant products. Sizes are indicated in bp. Complementarity and relative location of these sequences with the Star transcript sequence is indicated.

Article Snippet: Homologous sequences were compared against the murine cDNA derived from the longest Star mRNA (NM_011485) and analyzed using the Unigene database (NCBI), Blast2sequence (NCBI), and the Vector NTITM Suite 8 (InforMax, Inc.) software.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Nested PCR, Agarose Gel Electrophoresis, Clone Assay, Plasmid Preparation, Software, Generated, Amplification, Molecular Weight

Journal: PLoS ONE

Article Title: Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

doi: 10.1371/journal.pone.0022822

Figure Lengend Snippet: A . Total RNA was isolated from MA-10 cells and treated with DNase I. 3′ RACE was performed using two sequence-specific primers for PCR and nested PCR amplifications. Following agarose gel electrophoresis, bands were eluted, and cloned into the pGEM®-T Easy vector for sequencing. The results were analyzed by BLAST and Vector NTITM Suite 8 software. Left panel. Schematic diagram showing the relative location of the sequence-specific primers used. Right panel. Representative image of the nested PCR product generated. MW, DNA molecular weight ladder: -RT, no reverse transcriptase RT-PCR control. Arrows indicate the apparent sizes in bp. B . Total RNA extracted from MA-10 cells was treated with DNase or RNase prior to sequence-specific reverse transcription, followed by PCR/nested PCR and sequencing. Upper panel. Schematic diagram showing the RT primer (Rv.RT), PCR primers (Fw.1 and Rv.1), and nested PCR primers (Fw.2 and Rv.2) used in these experiments. A schematic representation of the full Star NAT sequence obtained after sequencing the PCR product is shown. Lower panel. Representative image of the RT-nested PCR product. +R, RNase-treated RNA control; -RT, no reverse transcriptase RT-PCR control; -p, RT reaction in the absence of primer; pi, RT reaction in the presence of a non-specific primer; MW, DNA molecular weight ladder. Arrows indicate apparent sizes in bp.

Article Snippet: Homologous sequences were compared against the murine cDNA derived from the longest Star mRNA (NM_011485) and analyzed using the Unigene database (NCBI), Blast2sequence (NCBI), and the Vector NTITM Suite 8 (InforMax, Inc.) software.

Techniques: Isolation, Sequencing, Nested PCR, Agarose Gel Electrophoresis, Clone Assay, Plasmid Preparation, Software, Generated, Molecular Weight, Reverse Transcription Polymerase Chain Reaction