Male and female mice were injected with dexamethasone (5 mg/kg; i.p.). Three hours later amygdala and NAc punches were collected for qPCR analysis. Total cellular RNA was extracted from amygdala and NAc punches using Qiazol reagent and the RNAeasy Midi kit (QIAGEN, Valencia, CA, United States). Total RNA was reverse transcribed into cDNA using VILO master mix (Invitrogen, Carlsbad, CA, United States). qPCR was performed in triplicate aliquots from each individual animal with Power SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, United States), 25 ng of cDNA and 0.5 μM of primers using an ABI Prism 7900HT (Thermo Fisher Scientific, Waltham, MA, United States) in the qPCR CoRE at Icahn School of Medicine at Mount Sinai. Primer sequences for GPR83, proSAAS and GAPDH are the same as used previously (Fakira et al., 2019 (link)). The primer sequences used for qPCR are: GAPDH Forward: 5′-TGAAGGTCGGTGTGAACG Reverse: 5′-CAATCTCCACTTTGCCACTG, GPR83 Forward: 5′-GCAGTGAGATGCTTGGGTTC Reverse: 5′-CCCACCAAT AGTATGGCTCA, and proSAAS: Forward: 5′-AGTGTATG ATGATGGCCC Reverse: 5′-CCCTAGCAAGTACCTCAG. The CT values for the technical replicates were averaged and analysis performed using the ΔΔCT method and normalized to saline controls. In some cases, qPCR reactions were repeated to determine the reliability of the primers and RNA samples.
Vilo master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
VILO master mix is a reagent used for the reverse transcription of RNA to cDNA. It contains all the necessary components for the conversion of RNA to complementary DNA, including reverse transcriptase enzyme, random primers, and buffer solutions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rnaeasy midi kit, by Qiagen (2 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Ezdnase, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Superscript 4, by Thermo Fisher Scientific (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Hs03044422 g1, by Thermo Fisher Scientific (2 mentions)
Human control rna, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr machine, by Thermo Fisher Scientific (2 mentions)
23 protocols using vilo master mix
1
Dexamethasone-Induced Gene Expression in Amygdala and NAc
2
RNA Extraction and qPCR Analysis in THP1 Cells
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RNA was extracted from THP1 cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA was treated with DNase I (Ambion) to remove contaminating DNA, and samples of ~1 μg of RNA were reverse transcribed using Superscript III or VILO Mastermix (Invitrogen). qPCR was performed using SYBR Green mix or SsoAdvanced SYBR Green mix (Bio‐Rad). The fold change in gene expression was calculated by the ΔΔCt method using glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as an internal control. The primers used for qPCR are listed in Table EV2 .
3
Profiling Gastric Epithelial Cell Development
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HS or FS epithelial cells were isolated from E14.5 or E18.5 HS or FS,73 (link) and total RNA from cells was DNase-treated as in previous studies or with EZ-DNase (Invitrogen, Waltham, MA).73 (link),74 (link) MMLV reverse transcriptase (Invitrogen) or VILO master mix (Invitrogen) were used to synthesize complementary DNA. TaqMan assays (Table 3 ) were used for qRT-PCR with TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA). For each gene assayed, at least 3 biological replicates per genotype were assayed in 3 independent experiments. Gapdh was used to normalize. For each target, expression units were calculated using the formula [2(–dCq)]×1000.15 (link) Error bars show SEM. Statistical tests used were either a Student’s t test or 1-way analysis of variance, as appropriate. Figure legends specify which statistical tests were used for each analysis.
TaqMan Assay Identifiers
| Gene | TaqMan ID |
|---|---|
| Atp4a | Mm00444417_m1 |
| Cldn10 | Mm01226326_g1 |
| Cldn18 | Mm00517321_m1 |
| Gata4 | Mm00484689_m1 |
| Gata6 | Mm00802632_m1 |
| Gif | Mm00433596_m1 |
| Hnf4a | Mm00433964_m1 |
| Krt15 | Mm00492972_m1 |
| Krt17 | Mm00495207_m1 |
| Krt23 | Mm00840789_m1 |
| Mist1 | Mm00627532_s1 |
| Muc5ac | Mm01276718_m1 |
| Muc6 | Mm00725165_m1 |
| Pgc | Mm00482488_m1 |
| Tff1 | Mm00436945_m1 |
| Tff2 | Mm00447491_m1 |
| Trp63 | Mm00495793_m1 |
| Gapdh | 4351309 |
4
Quantifying mRNA Expression in Tissues
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RT-PCR was used to analyze mRNA expression. Briefly, total RNA was extracted from lungs and human leukocytes using a Qiagen miRNEasy kit (catalog no.: # 217004). The quality and concentration of the input RNA were measured on the Synergy HT Take3 Microplate Reader (BioTek) and complementary DNA was prepared using SuperScript IV. VILO Master Mix (catalog no.: # 11756500, Invitrogen) for mRNA. Quantitative PCR (qPCR) was performed in duplicate using TaqMan Fast Advanced Master Mix (catalog no.: # 44-445-57) for mRNA using a Mx3000p Real-Time PCR System (Stratagene). The primers for the qPCR were purchased from Thermo Fisher Scientific/TaqMan. mRNA levels were normalized to internal control Tuba1a, and relative mRNA expression was reported.
5
Total RNA Isolation and cDNA Synthesis
6
Quantitative Assessment of mRNA Integrity
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RNA was quantified by Qubit and Nanodrop 1000 (both Thermo Fisher Scientific). An aliquot of 8 μL of extracted RNA was reverse transcribed with SuperScript IV and VILO master mix (Invitrogen) primed with random hexamers. To assess integrity and yield of human mRNA, transcripts for a human housekeeping gene (actin gamma 1, ACTG1, Hs03044422_g1) and bacterial 16S rRNA (16S, Ba04230899_s1, both Thermo Fisher Scientific) were quantified by quantitative reverse transcriptase PCR (RT-qPCR), TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), and a QuantStudio 3 real-time thermocycler (Thermo Fisher Scientific). Amplification conditions were 50°C for 2 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. Commercial human RNA (Human Control RNA, Thermo Fisher Scientific) and total RNA extracted from a pure culture of Escherichia coli (DH5α) were used as standards. A ratio of ACTG1 to 16S was calculated and compared to quality transcriptomic data.
7
Quantifying RNA Integrity and Yield
8
Profiling Circular RNA Biomarkers
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The primers for RT-PCR were designed according to the sequences of circRNA junction sites. Cell-free RNAs from CSF were extracted using Norgen Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Slurry Format) according to the manufacturer’s instructions. cDNAs were synthesized using the SuperScript VILO Master Mix and used as templates for PCR amplification using Invitrogen™ Platinum™ SuperFi™ PCR Master Mix. Then the PCR products were subjected to agarose gel electrophoresis and PCR product libraries were constructed with cDNA segments extracted from gel using KAPA Hyper Prep C Kit according to the manufacturer’s instructions. Lastly, NOVAseq NGS was performed and the sequencing results were compared to the sequence of each circRNA junction site respectively.
9
cDNA Synthesis from Total RNA
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The RNA that was acquired underwent reverse transcription by cDNA synthesis using superscript IV VILO master mix (Invitrogen by Thermo Fisher Scientific, Karnataka, India) in accordance with the guidelines provided by the manufacturer. Each reaction mixture consisted of total RNA (10 μL), RT buffer for M-MuLV (4 μL), 10x solution for M-MuLV (2 μL), M-MuLV reverse transcriptase (RNase H) (1 μL), ribonuclease inhibitor (0.5 μL), 10 mM dNTP mix (2 μL), and molecular grade water to make a final volume of 20 μL. The synthesized cDNA was stored at −20 °C until further use.
10
GPR83 Gene Expression Analysis
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