RNA was isolated from the hypothalamus (dams and offspring) using the AllPrep DNA/RNA Mini Kit (Qiagen) and reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR applications (Invitrogen). RNA quality was determined to be within accepted parameters using a NanoDrop spectrophotometer. Quantitative RT-PCR was performed with 1 µl of cDNA using an ABI 7500 Fast Thermal Cycler and the Fast SYBR Green Master Mix reagent (Applied Biosystems). All primer probes (Sigma-Aldrich; see electronic supplementary material, table S1) were designed to span exon boundaries ensuring amplification of only mRNA. For each gene, CT values were normalized to cyclophillin A (endogenous control, [30 (link)]. Relative expression values were obtained by the ΔΔCT method [31 (link)].
Superscript 3 first strand synthesis system for rt pcr applications
Manufactured by Thermo Fisher Scientific
The SuperScript III First-Strand Synthesis System is a reverse transcription (RT) kit designed for complementary DNA (cDNA) synthesis from RNA templates. It provides a streamlined workflow for the generation of first-strand cDNA from total RNA or messenger RNA (mRNA) for subsequent use in real-time PCR (RT-PCR) applications.
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2 protocols using superscript 3 first strand synthesis system for rt pcr applications
1
Hypothalamic Gene Expression Analysis
2
Quantitative RT-PCR of Hypothalamic Gene Expression
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RNA was isolated from the PN6 and adult hypothalamus of male and female PS and PC offspring using the AllPrep DNA/RNA Mini Kit (Qiagen) and reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR applications (Invitrogen). Quantitative RT-PCR was performed with 1μl of cDNA using an ABI 7500 Fast Thermal Cycler and the Fast SYBR Green Master Mix reagent (Applied Biosystems). Primer probes (Sigma-Aldrich) were designed to span exon boundaries ensuring amplification of only mRNA (see Table S1). For each gene, CT values were normalized to cyclophillin A (endogenous control). Relative expression values were obtained by the DDCT method calculated relative to control group.
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