The entire hypothalamus of late pregnant NestinΔGHR (n = 8) and control (n = 7) mice was collected and RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA), followed by incubation in DNase I RNase-free (Roche Applied Science) and then reverse transcription using 2 μg of total RNA, SuperScript II Reverse Transcriptase (Invitrogen) and random primers p(dN)6 (Roche Applied Science). Real-time PCR was performed using the 7500TM Real-Time PCR System (Applied Biosystems, Warrington, UK), Power SYBR Green Gene Expression PCR Master Mix (Applied Biosystems) and specific primers for target genes: Actb (forward: gctccggcatgtgcaaag; reverse: catcacaccctggtgccta), Gapdh (forward: gggtcccagcttaggttcat; reverse: tacggccaaatccgttcaca), Ghr (forward: atcaatccaagcctggggac; reverse: acagctgaatagatcctgggg), Stat5a (forward: cgctggactccatgcttctc; reverse: gacgtgggctcctcacactga) and Stat5b (forward: ggactccgtccttgataccg; reverse: tccatcgtgtcttccagatcg). Data were normalized to the geometric average of Actb and Gapdh. Relative quantification of mRNA was calculated by 2−ΔΔCt.
7500tm real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The 7500TM Real-Time PCR System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It enables the detection and quantification of targeted DNA or RNA sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Rnase free dnase 1, by Roche (3 mentions)
Random p dn 6 primers, by Roche (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Power sybr green gene expression pcr master mix, by Thermo Fisher Scientific (2 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using 7500tm real time pcr system
1
Quantifying Hypothalamic Gene Expression in Pregnant Mice
2
Hypothalamic Gene Expression Analysis
3
Hypothalamic RNA Extraction and Analysis
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Total RNA from the hypothalamus was extracted with TRIzol reagent (Invitrogen). Assessment of RNA quantity and quality was performed with an Epoch Microplate Spectrophotometer (Biotek). Total RNA was incubated with DNase I RNase-free (Roche Applied Science). Reverse transcription was performed with 2 µg of total RNA with SuperScript II Reverse Transcriptase (Invitrogen) and random primers p(dN)6 (Roche Applied Science). Real-time polymerase chain reaction was performed using the 7500TM Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). Relative quantification of mRNA was calculated by 2-ΔΔCt. Data were normalized to the geometric average of Actb, Gapdh and Ppia and reported as fold changes compared to values obtained from the control group (set at 1.0). The list of primers is available as Figure 3—source data 2 .
4
Hypothalamic Gene Expression in Leptin-Deficient Mice
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In this study, 7-or 12-day-old male and female Lep ob/ob mice and lean littermates fed ad libitum were decapitated, and the entire hypothalamus was collected. To evaluate the effects of fasting and leptin replacement, 10-day-old C57BL/6 mice were subjected to an overnight fast (16 h). At the moment of separation from the dams, the pups received a s.c. injection of either PBS or leptin (10 µg/g of body weight). The following morning, the pups received a second injection of PBS or leptin and were sacrificed 3 h later. A group of 10-day-old C57BL/6 mice fed ad libitum also received PBS injections at the same time and were used as controls. The hypothalamus was collected to determine the gene expression. Total RNA was extracted with TRIzol (Invitrogen). Real-time PCR was performed using the 7500TM Real-Time PCR System (Applied Biosystems), Power SYBR Green Gene Expression PCR Master Mix (Applied Biosystems) and specific primers for target genes (Table 1). Data were normalized to the geometric average of Actb, Gapdh and Ppia.
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