Mice were euthanized by overdose with isoflurane and the brains were quickly dissected out and frozen at −80 °C. The frozen mouse brains were then sectioned with a cryostat at −20 °C and loaded onto the PAN membrane frame slide (Applied Biosystems, Foster City, CA). The mDANs were dissected by the LCM with the ArturusXT micro-dissection system (Applied Biosystems) based on the presence of green fluorescent proteins (GFP) as described previously7 . Briefly, the total RNA was extracted from around 800 isolated mDANs per mouse brain with the PicoPure Isolation kit (Applied Biosystems) and the cDNAs were synthesized with the First Strand kit (QIAGEN, Valencia, CA) from equal amounts of total RNAs in the comparison groups. The SYBR Green real-time PCR detection method was used to quantitate the calcium channel expression. All the primers used in the qPCR were from QIAGEN and tested by the manufacturer. The expression levels were calculated as fold changes normalized with the β-actin. Data were statistically compared with unpaired t test, two tailed.
Picopure isolation kit
Manufactured by Thermo Fisher Scientific
The PicoPure Isolation kit is a lab equipment product designed for isolating high-quality RNA from small samples. It provides a reliable and efficient method for extracting RNA from limited tissue samples or single cells.
Automatically generated - may contain errors
Lab products found in correlation
Pan membrane frame slide, by Thermo Fisher Scientific (2 mentions)
Arturusxt micro dissection system, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Qiagen (2 mentions)
Sybr green 1 master mix, by Roche (2 mentions)
4 protocols using picopure isolation kit
1
Quantitative Analysis of Calcium Channel Expression in Mouse Dopaminergic Neurons
2
Quantifying Gene Expression in Mouse Brain
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Mouse brains of different genotypes and treatments were quickly dissected out and frozen down at −80 °C. The frozen mouse brains were sectioned with a cryostat at −20 °C and loaded onto the PAN membrane frame slide (Applied Biosystems, Foster City, CA). The dorsal striatum was dissected by the LCM with the ArturusXT micro-dissection system (Applied Biosystems) based on the anatomic landmarks in the striatum such as corpus callosum (CC), lateral ventricle (LV) and nucleus accumbens (Acb). The total RNAs were extracted with the PicoPure Isolation kit (Applied Biosystems) and the cDNAs were synthesized with the First Strand kit (QIAGEN, Valencia, CA) from equal amounts of total RNAs in the comparison groups. The SYBR Green real-time PCR detection method was used to quantify the Aldh1a1 and opioid receptors as well as the peptides. All the primers used in the qPCR were from QIAGEN and tested by the manufacturer. Gene expression was calculated as fold change normalized to β-actin.
3
Quantitative PCR for Stemness Genes
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Samples were collected via centrifugation and RNA isolated using PicoPure Isolation kit (Life Technologies) with DNase I (Qiagen) per manufacturer instructions. cDNA was generated from 1 μg RNA via SuperScript III First-Strand Synthesis kit (Life Technologies). Real-time qPCR was performed using custom designed primers and SYBR Green I Master mix (Roche) on a Lightcycler 480. Expression levels were normalized to GAPDH expression. Primer sequences used to detect endogenous expression of KLF4 and SOX2 were previously published38 (link). Custom designed primer sequences used are available upon request.
4
Quantitative PCR for Stemness Genes
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