RNA was extracted from mock-infected and ZIKV-infected (HS-2015-BA-01) primary neurons, at 6 and 24 hours post infection (hpi) in triplicates. RNA isolated from transfected Neuro-2A cells at 48 hours was obtained in triplicates as well. The RNA extraction was carried out using TRIzol reagent (Invitrogen) and precipitated with anhydrous ethanol as described [50 (link),55 (link)]. Total RNA resuspended in Ultrapure DNase/RNase-free distilled water (Invitrogen) was purified through miRNeasy mini kit columns (QIAGEN). RNase-free DNase set (QIAGEN) was added onto the columns to eliminate any trace of DNA. Total RNA was collected from the column using 50 μl of Ultrapure water and sent to Genome Québec, Montréal, Canada (https://cesgq.com/home ) for microarray analysis. The RNA concentration was measured with a spectrophotometer/fluorometer (DeNovix, DS-11 FX+).
Mirneasy mini kit columns
Manufactured by Qiagen
Sourced in Germany
The MiRNeasy mini kit columns are designed for the purification of total RNA, including small RNAs such as miRNA, from a variety of sample types. The columns use a silica-based membrane technology to efficiently capture and purify RNA molecules of all sizes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Qiazol reagent, by Qiagen (2 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr master mix, by Qiagen (1 mentions)
Miscript cdna synthesis kit, by Qiagen (1 mentions)
Cfx96 thermal cycler, by Bio-Rad (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Mini beadbeater 24, by Biospec (1 mentions)
Miscript sybr green qpcr mastermix, by Qiagen (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Iscript reverse transcription supermix kit, by Bio-Rad (1 mentions)
9 protocols using mirneasy mini kit columns
1
RNA Extraction and Microarray Analysis of ZIKV-Infected Neurons
2
Transcriptome Analysis of Clp1 Mutant Mice
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RNA was prepared from individual spinal cords from P14 wild-type and Clp1R140H/− mice using TRIzol and miRNeasy Mini kit columns (Qiagen) with on-column DNase digestion. Three RNA-seq libraries per genotype were made with the Kapa Stranded mRNA-Seq Kit in accordance with the manufacturer’s instructions; 100-nt paired-end reads were generated on a NovaSeq 6000.
3
Liver miRNA Extraction and Quantification
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Total miRNA was isolated from 30 mg liver tissue by homogenization in 700 μl Qiazol reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions and purified with the use of miRNeasy mini kit columns (Qiagen, Valencia, CA). Purified miRNA (1μg) was converted to cDNA using miScript cDNA synthesis kit (Qiagen, Valencia, CA) following the manufacturer’s standard protocol. Quantitative real-time PCR was performed with the gene specific primers using miScript SYBR Green PCR master mix (Qiagen, Valencia, CA) and CFX96 thermal cycler (Bio-rad, Hercules, CA). Threshold Cycle (Ct) values for the selected genes were normalized against RNU6-2 (internal expression control) values in the same sample.
4
Streptococcus uberis Mammary Gland RNA Extraction
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Only the rear right quarter (challenged with the Strep. uberis inoculum) was used for mammary gland RNA extraction and subsequent analysis. Tissue was weighted (~ 0.05 g), immediately placed in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) (1 mL) and homogenized using a Mini-Beadbeater-24 (Biospec Products Inc., Bartlesville, OK, USA) with two 30 s cycles, and 1 min incubation on ice in between the cycles. Samples were then centrifuged for 10 min at 12,000×g and 4 °C, and the supernatant was transferred to a separate tube and mixed with chloroform (0.2 mL). After centrifugation for 15 min at 12,000×g and 4 °C, the aqueous phase was transferred to a new tube, mixed with 100% ethanol (0.75 mL), and total RNA was cleaned using miRNeasy mini kit columns (Qiagen, Hilden, Germany) following manufacturer’s protocols. During purification, genomic DNA was removed using the RNase-Free DNase Set (Qiagen, Hilden, Germany). Quantity was determined using a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA), while integrity was assessed via a Fragment Analyzer™ (Agilent Technologies, Santa Clara, CA, USA). All samples had an RQN (RNA quality number) score greater than 7.0. RNA samples were stored at − 80 °C until analysis.
5
Extraction and Purification of Colon-Derived RNA
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An overall 20 g of fecal samples was taken from each candidate over a month. Using a swab, the samples were collected from either the stools’ mucinous region, as a rich source of colonocytes,10 or non‐mucinous areas, for evaluating the entire colon status. The collections were immediately dissolved in RNALater buffer (2 mL/g) and stored at −80°C for future analysis. One millilitre of patient dissolved stool was mixed with 3 mL of buffer containing 10 mmol/L Tris HCl (pH 7.4), 200 mmol/L NaCl, and 1 mmol/L EDTA and vortexed vigorously for 3 minutes. The mixture was centrifuged for 5 minutes at 12 000 g. The supernatants were transferred to the miRNeasy Mini Kit columns (QIAGEN) and preceded according to the manufacturer's protocol. To avoid genomic DNA contamination, RNA samples were treated with DNase I for 1 hour and examined by 1% agarose gel electrophoresis to evaluate RNA integrity. Additionally, the RNA concentration was estimated using the NanoDrop® ND‐1000 spectrophotometer (Thermo Fisher Scientific). The RNA purity was evaluated according to the A260/A280 ratio.
6
Liver miRNA Profiling from Biopsies
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Liver total RNA from biopsied tissue was isolated using Qiazol Reagent and miRNeasy Mini kit columns (Qiagen). RNA was quantified using a NanoDrop-1000 Spectrophotometer and purity was determined by calculating the 260/280 nm and 260/230 nm ratios. RNA integrity was assessed using the Agilent 2100 Bioanalyzer. For the selected (see above) miRNA screening, cDNA was synthetized from total RNA (1000 ng) using miScript® II RT Kit (Qiagen), following the manufacturer’s protocol. miRNA quantification was performed by real time PCR (qRT-PCR) using the miScript SYBR®Green qPCR Master Mix (Qiagen) on a 7900HT fast real time PCR system (Applied Biosystems) using a 384-well plate format. Reactions were performed in a final volume of 10 µl following these cycling conditions: 15 min at 95 °C for one cycle, then 40 cycles at 94 °C for 15 s and 58 °C for 30 s. The dissociation curve was analyzed at 95 °C for 15 s, followed by one cycle with increasing temperature starting at 60 °C and ending at 95 °C. RNU6 and RNU1A1 were used as reference small RNAs for normalization. miRNA relative expression analysis was performed using the GenEx software (MultiD Analyses AB, Sweden).
7
RNA Analysis of EV Fractions
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RNA analysis was performed from each “pellet” sample, fraction 2 (pre-EVs), fraction 3 (EV fraction), combined fractions 4 + 5 (middle fractions) and combined fractions 6–8 (protein fractions). Qiazol-sample mixes were defrosted on ice, combined with 0.2 volume of chloroform and left for 5 min at RT. Solutions were centrifuged (12,000× g, 15 min, 5 °C), to allow separation and collection of the aqueous phase, which was in turn combined with 2.5 volume of 96% ethanol. The sample mix was added to miRNeasy Mini Kit columns (Qiagen, Hilden, Germany, #217004) and washed according to the manufacturer’s instructions. Finally, the total RNA was eluted in 30 μL of nuclease free water. The small RNA profile of isolated RNAs was measured from 1 μL of sample using the Agilent Bioanalyzer Small RNA kit (#5067-1548, Agilent Technologies, Santa Clara, CA, USA). The concentration of 10–40 nt and 40–80 nt small RNA were calculated using the 2100 Expert software with custom ranges.
8
Plasma RNA Extraction Protocol
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Example 2
EDTA blood samples were collected from cases and control individuals and processed for plasma within 2 hours of collection. To avoid contamination with epithelial cells from the initial skin puncture the first blood tube collected during phlebotomy was not processed for plasma. Blood was centrifuged at 1300 g for 20 minutes at 10° C. The supernatant (plasma) was transferred into microcenlrifuge tubes followed by a second high-speed centrifugation step at 15500 g for 10 minutes at 10° C. to remove cell debris and fragments. The plasma was aliquoted into cryo vials, snap-frozen in liquid nitrogen and stored at −80° C. until use.
Total RNA (including miRNAs) was extracted from 400 μL of plasma. Denaturation and phase separation were conducted using TRIzol LS (Invitrogen, Germany) according to manufacturer's protocol, with a minor modification: 10 fmol of a C. elegans miR-39/miR-238 mixture was spiked-in. The aqueous phase was transferred into another tube, 1.5 volumes of absolute ethanol were added and the mixture was applied to miRNeasy Mini kit columns (Qiagen, Germany). After washing miRNAs were eluted in 30 μL of RNase-free water.
9
NMDA-Induced Retinal Transcriptome Analysis
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