Rnazol

Cell supernatant was collected and Maxwell RSC Viral Total Nucleic Acid purification kit was used to extract RNA from 250 μL of cell culture supernatants employing the Maxwell RSC Instrument (Promega, Madison, WI, USA). The remaining supernatant was conveniently stored at −80°C for the TCID₅₀ assessment. Each well was then thoroughly washed three times with pre-warmed PBS. Cells were lysed and collected in 100 μL of RNAzol (TEL-TEST, Inc., Friendswood, TX, USA). RNA extraction was performed employing the acid guanidium-phenol-chloroform (AGPC) extraction method, as elsewhere described (88 (link)). One μg of total RNA was reversed transcribed in a final volume of 20 μL using the Reverse Transcription Kit (Promega). Target cDNA was amplified by either ddPCR or rtPCR.

Cell supernatant was collected and Maxwell^® RSC Viral Total Nucleic Acid Purification Kit was used to extract RNA from 250 µL of cell culture supernatants employing the Maxwell^® RSC Instrument (Promega, Madison, WI, USA). The remaining supernatant was conveniently stored at −80 °C for the TCID50 assessment. Each well was then thoroughly washed three times with pre-warmed PBS. Cells were lysed and collected in 100 µL of RNAzol^® (TEL-TEST Inc., Friendswood, TX, USA). RNA extraction was performed employing the acid guanidium-phenol-chloroform (AGPC) extraction method, as elsewhere described [14 (link)]. One µg of total RNA was reversed transcribed in a final volume of 20 µL using the Reverse Transcription kit (Promega, Madison, WI, USA). Target cDNA was amplified by ddPCR.

To compare the effects of structurally related SCFA on endogenous TH gene expression, PC12 cells were treated with low concentrations of SCFA (PPA, BA, and VPA) or with vehicle for 48 hours. Total RNA was isolated from individual petri dishes using RNazol according to the manufacturer's protocol (Tel-Test, Inc., Friends-Wood, TX). Northern blot analysis was performed as described previously [64] . After transfer to Gene Screen Plus membranes, the filters were hybridized consecutively to labeled rat cDNA probe for TH and a probe for 18S ribosomal RNA. Filters were washed under proper stringency and exposed for autoradiography, using Kodak Bio Max film (Rochester, NY). The autoradiographs were scanned and quantified by Bio-Rad Quantity One software and the abundance of TH mRNA was expressed relative to 18S ribosomal RNA levels. The results were presented as fold change compared with the control (vehicle treated) group on the same Northern blot.

The transcription levels of the lipid metabolism related genes were quantified with RT-qPCR as described in a previous study [32 (link)]. Briefly, total RNA of the subset groups was extracted with RNAzol (Tel-Test Inc., Friendswood, TX, USA) from 3T3-L1 adipocytes according to manufacturer’s guideline. Total RNA molecules were quantified using NanoDrop system (Shimadzu Biotech, Kyoto, Japan) and the synthesis of complement DNA using a mixture of total RNA (5 μg), oligo-dT primer (Invitrogen, Carlsbad, CA, USA), dNTP, and reverse transcriptase (Superscript II, Invitrogen) was conducted using T100 ™ thermal cycler (Bio-Rad, Hercules, CA, US). With the synthesized cDNA template, qPCR was conducted using 2× Power SYBR Green (Toyobo Co., Osaka, Japan) with the following cycles: 15 s at 95 °C, 30 s at 55 °C, and 60 s at 70 °C. The primer sequences for target gene expression identification are stated as table (Supplementary Table S1). The reaction cycle during which the PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered the threshold cycle (Ct). The expression of the target gene was quantified relative to that of the housekeeping gene β-actin, based on a comparison of the Ct values at a constant fluorescence intensity according to Livak and Schmittgen’s method [33 (link)].