We purified total RNA from mouse quadriceps muscles by guanidinium thiocyanate-phenol-chloroform extraction. Briefly, we homogenized tissues in TRIsure (Bioline BIO-38033) and mixed with 1-bromo-3-chloropropane. After centrifugation, we mixed aqueous phase with 2-propanol to precipitate RNA. The precipitate was pelleted by centrifugation and washed with ethanol. We analyzed RNA with NanoDrop 2000 (Thermo) to determine purity and quantity and used 1 µg of RNA for first-strand cDNA synthesis (Quanta 95048). We performed quantitative RT-PCR using SensiFast SYBR green (Bioline) on the CFX384 Real Time PCR system (BioRAD). We expressed data as relative expression of Pdk4 mRNA by comparative threshold method using 36B4 as the internal control. We used the following primers: Pdk4 forward 5′-CCGCTGTCCATGAAGCA-3′, reverse 5′-GCAGAAAAGCAAAGGACGTT-3′ and 36B4 forward 5′-GCAGACAACGTGGGCTCCAAGCAGAT-3′, reverse 5′-GGTCCTCCTTGGTGAACACGAAGCCC-3′.
Bio 38033
Manufactured by Meridian Bioscience
The BIO-38033 is a laboratory equipment product manufactured by Meridian Bioscience. It is designed for performing routine laboratory tasks. The product's core function is to assist in the preparation and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Sensifast sybr green, by Meridian Bioscience (1 mentions)
Cfx384 real time pcr system, by Bio-Rad (1 mentions)
M mulv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Truseq small rna sample kit, by Illumina (1 mentions)
Rp8092h, by Illumina (1 mentions)
3 protocols using bio 38033
1
Quantifying Mouse Muscle Pdk4 mRNA
2
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from ST2 cells using TRIsure (Bioline, BIO-38033). Medium was aspirated and 1000 μl of TRIsure was added to 6-wells. To separate RNA from DNA and proteins, 200 μl of chloroform (Carl Roth, 6340.1) was added. To precipitate RNA from the aqueous phase, 400 μl of 100% isopropanol (Carl Roth, 6752.4) was added and RNA was incubated at −20°C overnight. cDNA synthesis was done using 1 μg of total RNA, 0.5 mM dNTPs (ThermoFisher Scientific, R0181), 2.5 μM oligo dT-primer (Eurofins MWG GmbH, Germany), 1 U/μl Ribolock RNase inhibitor (ThermoFisher Scientific, EO0381) and 1 U/μl M-MulV Reverse transcriptase (ThermoFisher Scientific, EP0352) for 1 h at 37°C or 5 U/ μl RevertAid Reverse transcriptase for 1 h at 42°C. The PCR reaction was stopped by incubating samples at 70°C for 10 min.
3
Small RNA Sequencing of Synchronized Worms
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Synchronised animals were grown to 1 day-old adults 20 °C. After being washed with M9 to remove bacteria, animals were resuspended in TRIsure (Bioline, BIO-38033). Animals were lysed with 5× freeze-thaw cycles in liquid nitrogen. Total RNA was isolated by chloroform extraction. For 5′ -independent libraries, 5 µg of total RNA was treated with 5′ polyphosphatase (Epicenter, RP8092H). Small RNAs were indexed using the TruSeq small RNA sample kit (Illumina) and size selected by gel separation in 6% TBE gels (Life Tech) and subsequently purified.
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