RNA extraction of 2.5 weeks above-ground tissues was conducted using Trizol. Reverse transcription was conducted using SuperScript III (Invitrogen), 5 μg RNA and random primers (Sigma) PCR amplification was conducted using GoTaq Green (Promega) with primers listed in Supplementary Table S8 .
Random primers
Manufactured by Merck Group
Sourced in United States
Random primers are short oligonucleotide sequences used in various molecular biology techniques. They serve as starting points for DNA synthesis, allowing the amplification of unknown or random DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Superscript 3, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Sybr green mastermix, by Eurogentec (2 mentions)
Stainless steel bead, by Qiagen (2 mentions)
Fibrous tissue mini kit, by Qiagen (2 mentions)
Fs buffer, by Thermo Fisher Scientific (2 mentions)
Chloroform, by Merck Group (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Tissuelyser lt, by Qiagen (2 mentions)
Uno 2, by Analytik Jena (2 mentions)
Abi prism 7700 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Omniscript rt kit, by Qiagen (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Tri reagent, by Merck Group (2 mentions)
7900ht fast real time system, by Thermo Fisher Scientific (2 mentions)
Amplitaq dna polymerase, by Thermo Fisher Scientific (2 mentions)
20 protocols using random primers
1
RNA Extraction and Reverse Transcription
2
Quantitative RT-PCR Analysis of Cerebellum Transcripts
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For RNA sequencing (RNA-Seq) analysis, cerebella from control male mice at different ages were isolated and maintained in RNA later stabilization reagent (76104, QIAGEN). Total RNA was extracted, and DNase treated using the RNAeasy Mini Kit (217004, QIAGEN). For validation assays, total RNA was isolated using the Trizol reagent (15596018, Invitrogen) according to the manufacturer’s protocol. Genomic was digested by RNase-free DNase (N2111, Roche). RNA purity and concentration were quantified using the NanoDrop 2000 UV spectrophotometer (Thermo Scientific). In all, 1 µg of total RNA was reverse-transcribed with random primers (11034731001, Sigma Aldrich) using M-MLV reverse transcriptase (M1705, Promega), according to the manufacturers’ instructions. 15 ng of cDNA were used as template for quantitative RT-PCR (qPCR) analysis. The qPCR reactions were performed using the LightCycler 480 System (Roche) with SYBR Green I Master Mix (04887352001, Roche) following the manufacturer’s instructions. The 2−ΔCt method was applied to calculate differences in the gene expression using L34 gene for data normalization. Primers (Sigma) used in this study are listed in Supplementary Table 1 . Male mice (n = 6 for both controls and VPA) were used for qPCR analyses.
3
Quantitative RT-PCR Analysis of BV-2 Cells
4
RNA-Binding Protein SRSF6 Interaction
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RNA immunoprecipitation of Tnc mRNA bound to SRSF6 protein was performed as described65 (link), with slight modifications. 30 μg pcDNA3.1-T7-SRSF6 or control plasmid was transiently transfected into NIH-3T3 cells in a 15-cm plate, and UV-crosslinking was performed at 400 mJ/cm2 using a Stratalinker UV Crosslinker (Stratagene). Immunoprecipitation was done using anti-T7 antibody (CSHL; mAb 42 1-87) coupled to Protein G-Dynabeads (Life Technologies); RNA was extracted from the immunoprecipitate, and cDNA was synthesized using random primers (Sigma-Aldrich). RT-PCR was performed using primers specific for each alternatively spliced Tnc isoform, i.e., to amplify across exons 9 and 10, or exons 15 and 16, respectively (for details of primer sequences, see Supplementary Information ), and 32P-radioactive PCR products were separated as described above. RNA CLIP qPCR was performed essentially as above, except that extracts were treated with micrococcal nuclease before immunoprecipitation using Protein G-Dynabeads coupled with either anti-T7 (CSHL; mAb 42 1-87) or control IgG1 (CSHL; AK105, against E. coli MBP) antibody. RNA enrichment was measured by RT-qPCR using exon-specific primers, and calculated as the relative levels in T7 antibody versus control IgG1 immunoprecipitates.
5
Transcriptomic Analysis of Plant Samples
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One hundred milligrams of plant material was collected and ground in liquid nitrogen with mortar and pestle. Total RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen) with an extra-protocol passage of 15 min of RNase-Free DNase (Qiagen) and resuspended in 30 μl of sterile nuclease-free water. RNA concentration was measured using a Nanodrop ND‐1000 spectrophotometer (Nano Drop Technologies Llc, Wilmington, Delaware, USA). First-strand cDNA synthesis was performed with a SuperScript-IV Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA), using 2 μg of RNA and 1 μl of random primers (Sigma). qRT-PCR was performed in plate using Taq® qPCR Master Mix (Promega) with SYBR Green technology in either QuantStudio 12K Flex or QuantStudio 5 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) instrument. The total volume of each reaction was 10 μl using 0.25 μl of primer mix (10 μmol). Sequences of the used primers are reported in Supplementary Table S1
6
Quantitative RT-PCR for Neural Lineage Markers
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Selection of candidate markers was based on the results from our previous work with cells of this type, along with potential relevance to the current study. Emphasis was placed on markers associated with immature cells of neural lineage, as well as markers for neural and glial differentiation. Constraints imposed by species specificity limit the individual markers available for work on porcine material. The gene-specific primers used in this study are shown in Supplemental Table 1 .
Two micrograms of total RNA from the sample preparation were reverse-transcribed with Omniscript® reverse transcriptase kit (Qiagen) and 10 µM random primers (Sigma), according to the manufacturer’s instructions. Quantitative PCR was performed by using a 7500 Fast Real-Time PCR System (Applied Biosystems, Irvine, CA, USA) using Power SYBR Green (Applied Biosystems). Resolution of the product of interest from nonspecific product amplification was achieved by melting curve analysis. β-Actin was used as an endogenous control to normalize gene expression. The following real-time PCR protocol was used: denaturation program (95°C for 10 min), quantification program (95°C 15 sec and 60°C 1 min) repeated 40 cycles, melting curve program (95°C 15 s and 60°C 1 min with continuous fluorescence measurements), and finally a cooling program down to 40°C. Each reaction was performed in triplicate.
Two micrograms of total RNA from the sample preparation were reverse-transcribed with Omniscript® reverse transcriptase kit (Qiagen) and 10 µM random primers (Sigma), according to the manufacturer’s instructions. Quantitative PCR was performed by using a 7500 Fast Real-Time PCR System (Applied Biosystems, Irvine, CA, USA) using Power SYBR Green (Applied Biosystems). Resolution of the product of interest from nonspecific product amplification was achieved by melting curve analysis. β-Actin was used as an endogenous control to normalize gene expression. The following real-time PCR protocol was used: denaturation program (95°C for 10 min), quantification program (95°C 15 sec and 60°C 1 min) repeated 40 cycles, melting curve program (95°C 15 s and 60°C 1 min with continuous fluorescence measurements), and finally a cooling program down to 40°C. Each reaction was performed in triplicate.
7
Human and NHP Immune Gene Expression
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The total RNA extracted from the brain tissues of human and non-human primate (NHP) cohorts was used for cDNA synthesis using random primers (Sigma-Aldrich, St. Louis, MO, USA) and SuperScript II reverse transcriptase (ThermoFischer Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Using appropriate primers (Table S1 ), human and rhesus macaque immune gene transcripts were quantified and normalized to GAPDH and reported as the fold change relative to the control group (HIV[−]) for the human cohort and the SIV[+]/ART group for the NHP cohort [46 (link)].
8
RNA Extraction and cDNA Synthesis Protocol
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Tissue was quickly dissected and frozen on either dry ice or liquid nitrogen and stored at −80 °C. Tissue was homogenized in a Trizol reagent (QIAzol Lysis Reagent, Qiagen) using a stainless steel bead (Qiagen) and a TissueLyser LT (Qiagen) for 3 min at 20 Hz. Then, 200 μl chloroform (Sigma-Aldrich) was added and tubes were shaken vigorously for 15 sec and left at RT for 2 min, followed by centrifugation at 4 °C for 15 min at 12,000×g. The aqueous phase was mixed 1:1 with 70% ethanol and further processed using RNeasy Lipid Mini Kit following the instructions provided by the manufacturer. For muscle tissue, the lysis procedure, described the enclosed protocol in the Fibrous Tissue Mini Kit (Qiagen), was followed. After RNA extraction, RNA content was measured using a NanoDrop 2000 (Thermo Fisher) and 500 ng of RNA was converted into cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma-Aldrich) and incubated for 3 min at 70 °C followed by addition of dNTPs, RNase out, Superscript III (Thermo Fisher) and placed in a thermal cycler for 5 min at 25 °C, 60 min at 50 °C, 15 min at 70 °C, and kept at −20 °C until further processing.
9
RNA Extraction and Real-Time qRT-PCR
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Plant samples were collected and frozen in liquid nitrogen immediately. The samples were ground into fine power and total RNA was extracted from them using RNeasy plant mini kits (Qiagen, http://www.qiagen.com/ ). First-strand cDNA was synthesized using SuperScript III (Invitrogen, http://www.invitrogen.com/ ) with oligo(dT)17 primer plus random primers (sigma). Real-time qRT–PCR was performed based on the corresponding cDNA samples using gene-specific primers as shown in Supplemental Table 1 as described previously (Luo et al., 2007 (link)).
10
Quantitative Real-Time RT-PCR Analysis
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Half a microgram of total RNA was taken for cDNA synthesis using Omniscript RT Kit (Qiagen), random primers (4 μM, Sigma-Aldrich), oligo(dT) primer (1 μM, QBiogene Inc.), and RNase inhibitor (10 U, Fermentas). The reaction was performed in 20 μl of total volume, according to manufacturer’s protocol, using thermocycler UNO II (Biometra). The cDNA was diluted 10-fold and a 5-μl aliquot was taken for real-time PCR performed using Taqman 2× PCR Master Mix (Roche), Exiqon probe (100 nM), and appropriate primers (200 nM each; Data Sheet 1 in Supplementary Material) designed using dedicated software from the Roche web site. The reaction was carried out using ABI PRISM7700 Sequence Detection System (Applied Biosystems) and the following thermal conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, 1 min at 60°C, and 1 min at 72°C. The experiments were performed in triplicates. The relative amount of cDNA was calculated using comparative ΔCt method. ΔCt values of the samples of interest were compared with a calibrator (RNA of known concentration pooled from several samples). The Ct values of both the calibrator and the samples of interest were normalized to the expression of three control genes, ATP6V1, HADHA, and UBE2D2.
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