For quantitative reverse transcription-PCR (qRT-PCR), Total RNA was extracted from individual replicate samples using a Micro Scale RNA Isolation Kit (Ambion). RNA samples extracted from cholinergic neurons were reverse-transcribed into cDNA using a TaqMan Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using an Applied Biosystems 7500 Real-Time System and TaqMan assays (Applied Biosystems). Samples containing no reverse transcriptase were used as negative controls. Expression values are relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Samples were analyzed in triplicate. Student’s t-test was used for statistical analysis. For single-neuron RT-PCR, MHb slice neuron cytoplasm was aspirated into a recording patch pipette and the contents were expelled into a microcentrifuge tube containing 75% ice-cold ethanol and stored at −20°C for at least 2 h before single-cell RT-PCR experiments were done as previously described (Zhao-Shea et al., 2011 (link)). Primer sequences for single-neuron RT-PCR experiments are indicated in Table S2 .
Micro scale rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro Scale RNA Isolation Kit is a laboratory equipment product designed for the extraction and purification of small amounts of RNA from various sample types. It provides a reliable and efficient method for isolating high-quality RNA from minimal starting material.
Automatically generated - may contain errors
Lab products found in correlation
7900ht fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman primer, by Thermo Fisher Scientific (2 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Moflo cell sorter, by Beckman Coulter (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
7500 real time system, by Thermo Fisher Scientific (1 mentions)
Accudrop beads, by BD (1 mentions)
Eight peak rainbow beads, by Spherotech (1 mentions)
Influx cell sorter, by BD (1 mentions)
Taqman primer probe set, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
6 protocols using micro scale rna isolation kit
1
Single-cell qRT-PCR of Cholinergic Neurons
2
Quantifying Vasopressin Receptor Expression
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Populations of Venus‐positive cells (L‐cells) or Venus‐negative cells (non‐L‐cells) of purity >90% were separated from the tissues of GLU‐Venus mice using a MoFlo cell sorter (Beckman Coulter, High Wycombe, UK) as described previously (Reimann et al. 2008 ). RNA was extracted from FACS‐sorted cells using a microscale RNA isolation kit (Ambion, Austin, TX, USA) and was then reverse transcribed to cDNA in accordance with standard protocols. The appropriate amount of first‐strand cDNA template was mixed with specific TaqMan primers (Applied Biosystems, Foster City, CA, USA), water and PCR Master Mix (Applied Biosystems), and a quantitative RT‐PCR was conducted using a 7900HT Fast Real‐Time PCR system (Applied Biosystems). β‐actin was used as the normalization control. The primer/probe pairs used in the present study were: Avpr1a: Mm00444092_m1; Avpr1b: Mm01700416_m1; and Avpr2: Mm01193534_g1 (all from Applied Biosystems). All experiments were performed on at least three cDNAs each isolated from one mouse (n = 3 mice).
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA from FACS-sorted cells [37 (link)] was isolated using a Microscale RNA Isolation Kit (Ambion) and reverse transcribed according to standard protocols. Quantitative RT-PCR was performed with a 7900 HT Fast Real-Time PCR system (Applied Biosystems). The PCR reaction mix consisted of first-strand cDNA template, appropriate TaqMan probe/primer mix, and PCR Master Mix (Thermo Fisher Scientific). The expression of Scgn was compared with that of Actb measured on the same sample in parallel on the same plate, demonstrating a CT difference (ΔCT). The mean, standard error, and statistical analyses were performed on the ΔCT data and only converted to relative expression levels (2 ˆ ΔCT) for presentation in the figures.
The total RNA collected from cell lines using a Paris Kit (Thermo Fisher) was reverse-transcribed using 5X All-In-One Reverse Transcriptase MasterMix (Applied Biological Materials, Richmond, BC, Canada), and quantitative RT-PCR was conducted using a TaqMan Fast Mix Gene Expression Assay with primers (Thermo Fisher Scientific) as listed inTable S1 . Gene expression was calculated using the ΔΔCt method [42 (link)]. H3f3a (mGLUTag) and Hist1h3a (hNCI-H716) were used as control genes as they have been previously established to lack circadian rhythms [16 (link),17 ,21 (link)].
The total RNA collected from cell lines using a Paris Kit (Thermo Fisher) was reverse-transcribed using 5X All-In-One Reverse Transcriptase MasterMix (Applied Biological Materials, Richmond, BC, Canada), and quantitative RT-PCR was conducted using a TaqMan Fast Mix Gene Expression Assay with primers (Thermo Fisher Scientific) as listed in
4
Whole-Brain and Region-Specific RNA Isolation
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For whole-brain experiments (Supplemental Fig. 2), total RNA was isolated using a mirVana Kit
(Ambion). For region-specific experiments (Fig.
3 ), brains were removed and placed on glass slides and snap-frozen in liquid nitrogen. Serial
coronal sections (1 mm) were prepared and the PFC, NAc, and VTA were isolated using tissue punches
(Harris UNI-CORE, 1.0 mm; Electron Microscopy Sciences). Total RNA was isolated from the samples
using a Micro Scale RNA Isolation Kit (Ambion). miRNAs were reverse-transcribed using a miRNA TaqMan
Reverse Transcription Kit (Applied Biosystems). Quantitative reverse transcription-polymerase chain
reactions (qRT-PCR) were done using an Applied Biosystems 7500 Real-Time System and miRNA TaqMan
assays (Applied Biosystems). Samples containing no reverse transcriptase were used as negative
controls. Relative gene expression was calculated using the 2−ΔΔCtmethod (Livak and Schmittgen 2001 (link)). Expression
values are relative to sno202, a small nucleolar RNA not regulated by nicotine. Samples were
analyzed in triplicate. Student's t-test was used for statistical analysis.
(Ambion). For region-specific experiments (
3
coronal sections (1 mm) were prepared and the PFC, NAc, and VTA were isolated using tissue punches
(Harris UNI-CORE, 1.0 mm; Electron Microscopy Sciences). Total RNA was isolated from the samples
using a Micro Scale RNA Isolation Kit (Ambion). miRNAs were reverse-transcribed using a miRNA TaqMan
Reverse Transcription Kit (Applied Biosystems). Quantitative reverse transcription-polymerase chain
reactions (qRT-PCR) were done using an Applied Biosystems 7500 Real-Time System and miRNA TaqMan
assays (Applied Biosystems). Samples containing no reverse transcriptase were used as negative
controls. Relative gene expression was calculated using the 2−ΔΔCtmethod (Livak and Schmittgen 2001 (link)). Expression
values are relative to sno202, a small nucleolar RNA not regulated by nicotine. Samples were
analyzed in triplicate. Student's t-test was used for statistical analysis.
5
Isolation and Quantification of Venus-Expressing Cells
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Populations of Venus-positive cells (L cells) or Venus-negative cells (non-L cells) of purity greater than 90% were separated from the tissues of GLU-Venus mice using a BD Influx cell sorter running BD FACS Software as previously described (16 (link)). Laser alignment was performed using eight-peak rainbow beads (Spherotech), and drop delay was determined using BD Accudrop beads. RNA was extracted from FACS-sorted cells by a microscale RNA isolation kit (Ambion) and reverse transcribed to cDNA according to standard protocols. A first-strand cDNA template was mixed with specific TaqMan primers (Applied Biosystems), water, and PCR master mix (Applied Biosystems), and quantitative RT-PCR was conducted using a 7900HT Fast real-time PCR system (Applied Biosystems). β-Actin was used as the normalization control. The primer/probe pairs used in this study were from Applied Biosystems: Agtr1, Mm01957722_s1, and Mas1, Mm00434823_s1. All experiments were performed on at least three cDNAs isolated from one mouse each.
6
RNA and Protein Extraction from Tissues
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Tissues used for RNA and protein extraction were washed in PBS and placed in RNAlater (Ambion, Life Technologies, Paisley, UK) and frozen until processed. Samples were homogenized in Tri-reagent (Sigma), and then treated as below. Total RNA from FACS-sorted cells prepared from GLU-Venus transgenic mice was isolated using a micro scale RNA isolation kit (Ambion). All samples were reverse transcribed according to standard protocols. Quantitative RT-PCR was performed with 7900 HT Fast Real-Time PCR system (Applied Biosystems, Life Technologies), using verified Taqman primer/probe sets supplied by Applied Biosystems. In all cases expression was compared with that of β-actin measured on the same sample in parallel on the same plate, giving a CT difference (ΔCT) for β-actin minus the test gene. Mean, standard error, and statistics were performed on the ΔCT data and only converted to relative expression levels (2ΔCT) for presentation in the figures.
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