Material stored in RNAlater (ThermoFisher Scientific) was thawed on ice, diluted with 0.7 mL nuclease-free PBS and pelleted by centrifugation at 6 k × g for 5 min at 4°C. RNA was extracted from the resulting pellet using TRIzol (ThermoFisher Scientific) according to the manufacturer’s instructions. RNase-free glycogen (10 μg) was used as a carrier during the precipitation phase to improve RNA yield. RNA yield was measured using the Qubit RNA high sensitivity kit (ThermoFisher Scientific), and sample purity and integrity was verified by TapeStation (Agilent) electrophoresis using RNA ScreenTape. To process samples for metantranscriptomic sequencing, total RNA was depleted of rRNA using the QIAseq FastSelect 5S/16S/23S kit (beta version, Qiagen), sequencing libraries were generated using the SMART-Seq Stranded for total RNA-seq kit (Takara) and 75 base pair, paired end reads were generated using an Illumina MiSeq v3 platform.
Qubit rna high sensitivity kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit RNA High Sensitivity kit is a fluorometric-based assay designed to accurately quantify low concentrations of RNA. The kit uses a fluorescent dye that binds specifically to RNA, allowing for precise measurement of RNA content in a sample. The assay is sensitive and requires only a small sample volume, making it suitable for analyzing limited RNA samples.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Rna 6000 pico kit, by Agilent Technologies (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Tapestation system, by Agilent Technologies (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Rneasy plus minispin column kit, by Qiagen (2 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (2 mentions)
Qiaseq fastselect 5s 16s 23s kit, by Qiagen (1 mentions)
Smart seq stranded for total rna seq kit, by Takara Bio (1 mentions)
Miseq v3 platform, by Illumina (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qubit broad range rna kit, by Thermo Fisher Scientific (1 mentions)
Qubit high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
36 protocols using qubit rna high sensitivity kit
1
Metagenomic RNA Extraction and Sequencing
2
FFPE Nucleic Acid Extraction for Molecular Analysis
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For molecular analyses, DNA and RNA were isolated from FFPE-derived tumor tissue and the precancerous lesion if available. DNA was isolated, quantified, and precipitated manually as described before.14 (link) After precipitation, the final concentration was determined using the Qubit High Sensitivity Kit (Thermo Fisher Scientific, Waltham, MA).
RNA was isolated using the ReliaPrep FFPE Total RNA Miniprep System (Promega, Madison, WI) according to the manufacturer's protocol, omitting the DNase treatment step. RNA concentrations were measured with the Qubit RNA Broad Range Kit (Thermo Fisher); samples were diluted to a concentration of 30 ng/µl and measured again with the Qubit RNA High Sensitivity Kit (Thermo Fisher). Subsequently, 60 ng of either DNA or RNA was used as input for the library preparation.
RNA was isolated using the ReliaPrep FFPE Total RNA Miniprep System (Promega, Madison, WI) according to the manufacturer's protocol, omitting the DNase treatment step. RNA concentrations were measured with the Qubit RNA Broad Range Kit (Thermo Fisher); samples were diluted to a concentration of 30 ng/µl and measured again with the Qubit RNA High Sensitivity Kit (Thermo Fisher). Subsequently, 60 ng of either DNA or RNA was used as input for the library preparation.
3
RNA Isolation Using Trizol LS Reagent
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RNA was isolated using Trizol LS (TRI) reagent (Invitrogen, Carlsbad, CA, USA, cat. #: 10296028) according to the manufacturer’s instructions with minor adjustments. In brief, 100 µL SP and 300 µL TRI Reagent® were mixed and incubated for 5 min. Then, 80 µL chloroform was added, mixed, and incubated for 2–3 min. The sample was centrifuged at 12,000× g at 4 °C for 15 min. The upper colorless phase was transferred to a new tube where 200 µL isopropanol was added, mixed, and incubated for 10 min. The sample was then centrifuged at 12,000× g at 4 °C for 10 min and the supernatant removed. The pellet was washed with 400 µL 75% ethanol, vortexed and centrifuged at 7500× g at 4 °C for 5 min. The supernatant was discarded, and the sample was vacuum-dried. The RNA pellet was resuspended in 20 µL RNase-free water, incubated on a heat block at 58 °C for 15 min and stored at −80 °C.
The quality of the isolated RNA was checked with the Agilent small RNA Bioanalyzer kit (Agilent, Santa Clara, CA, USA) according to manufacturer’s instructions and RNA content was evaluated with the Qubit® RNA High Sensitivity kit (Thermo Fisher Scientific, Waltham, MA, USA) using 2–5 µL sample.
The quality of the isolated RNA was checked with the Agilent small RNA Bioanalyzer kit (Agilent, Santa Clara, CA, USA) according to manufacturer’s instructions and RNA content was evaluated with the Qubit® RNA High Sensitivity kit (Thermo Fisher Scientific, Waltham, MA, USA) using 2–5 µL sample.
4
Urinary RNA Extraction and Sequencing
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First‐morning urine samples were processed within 2 to 4 hours of receipt to preserve RNA. Fresh urine was aliquoted to 1 or 2 50‐mL centrifuge tubes, centrifuged at 1000g, for 10 minutes at 4 °C. The supernatant was discarded, and the pellet was washed with 10‐mL ice‐cold PBS and centrifuged at 1000g for 10 minutes at 4 °C. The supernatant was discarded, and the cell pellet was resuspended in 1‐mL cold PBS and transferred to a 1.5‐mL Eppendorf tube and centrifuged at 1000g for 10 minutes at 4 °C. The supernatant was removed, and the cell was lysed with 350‐μL RLT Buffer with β‐ME from a Qiagen RNA extraction kit (74104). The pellet was broken by pipetting up and down and later centrifuged at 16000g for 5 minutes at 4 °C. We then followed the Qiagen RNA extraction kit protocol to store RNA for measurement or library preparation. Qubit RNA High Sensitivity kit (Thermo Fisher Q32852) was used to quantify RNA. The minimum amount of RNA required to generate a library was 5 ng. The low centrifugation speed ensured that the exosomes remained in the solution and were therefore excluded from analysis. Full‐coverage transcriptomes were obtained by RNAseq using an Ion Torrent Gene Studio Platform (Thermo Fisher) with quantifications of 16 000 to 19 000 mRNA transcripts per sample. Data were normalized before statistical analysis.
5
RNA Extraction and Sequencing Protocol
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Total RNAs were extracted from sorted cell suspensions using TRIzol LS Reagent (Invitrogen,10296010) following the manufacturer’s instructions. RNA concentration was determined using the Qubit RNA High Sensitivity Kit (ThermoFisher, Q32855) and integrity was assessed using RNA 6000 Pico Kit (Agilent, 5067-1513) run on the Agilent 2100 Bioanalyzer. 100ng-400ng samples with RNA Integrity Number (RIN) > 9 were submitted to MedGenome for library preparation using the Illumina TruSeq Stranded mRNA Library Kit and PE100 sequencing on NovaSeq.
6
Oocyst RNA Extraction for Sequencing
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Oocysts frozen in TRIzol were thawed on ice and 2 mL was transferred into a glass homogenizing tube. The suspension was ground using a Teflon pestle for 3–4 cycles of 25 grinds each. The samples were periodically examined using microscopy to monitor the progress of oocyst and sporocyst breakage. Homogenates were transferred into 2 mL RNAse-free microcentrifuge tubes, mixed with chloroform, and incubated at room temperature for 3 min. The tubes were centrifuged at 13,500× g for 15 min at 4 °C. The upper aqueous phase was transferred into new tubes and mixed with an equal volume of 70% ethanol. The total RNA was then isolated using a RNeasy Kit (QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. The total RNA was quantified using a Qubit 3.0 fluorometer and Qubit RNA High Sensitivity kit (Thermo Fisher Scientific) and the quality was assessed using a Bioanalyzer 2100 and RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). The RNA samples were frozen at −80 °C. The total RNA was processed using an Invitrogen Turbo DNA-free kit (Thermo Fisher Scientific). The total RNA of suitable quality for RNA-Seq had an RNA Integrity Number (RIN) ≥ 7 when assessed using a Bioanalyzer 2100.
7
RNA Extraction for Notch Transcriptome
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Total RNA from cell lines used to establish the Notch transcriptomic signature was isolated using the RNeasy Mini kit (cat #74104, Qiagen) according to the manufacturer’s instructions. RNA concentration was determined using Qubit RNA High sensitivity kit (cat #Q32852, Thermo Fisher Scientific), and RNA integrity score was determined using Bioanalyzer RNA 6000 Nano Kit (cat # 5067–1511, Agilent Technologies) for at least one sample per group. All samples were in addition controlled for quality at the NGI facility at the SciLifeLab laboratory (Stockholm, Sweden), and samples that failed the internal reception control were excluded from library preparation.
8
RNA Extraction from T. vaginalis Cells
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RNA was extracted from T. vaginalis isogenic strains. For each condition, two aliquots of 2 × 106 exponentially growing cells have been harvested, washed and resuspended in 700 μL of RNAlater (ThermoFisher Scientific, Waltham, MA, USA), then stored at −80 °C until use.
Samples were then thawed on ice, diluted with 0.7 mL nuclease-free PBS, and pelleted by centrifugation at 6 k× g for 5 min at 4 °C. RNA was extracted from the resulting pellet using TRIzol (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, with some modifications. Briefly, TRIzol and chloroform were used to lyse cells and solubilise cell components, then RNA was precipitated, washed, and resuspended in 30 µL nuclease-free water.
RNA concentration was assessed by Qubit RNA High Sensitivity kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. UV absorbance at 230 nm, 260 nm, and 280 nm was measured using a nanodrop 2000 c spectrophotometer as an indicator of purity. RNA integrity and absence of genomic DNA contamination were confirmed using a TapeStation System (Agilent, Santa Clara, CA, USA) with the resulting gel images and electropherograms manually examined.
Samples were then thawed on ice, diluted with 0.7 mL nuclease-free PBS, and pelleted by centrifugation at 6 k× g for 5 min at 4 °C. RNA was extracted from the resulting pellet using TRIzol (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, with some modifications. Briefly, TRIzol and chloroform were used to lyse cells and solubilise cell components, then RNA was precipitated, washed, and resuspended in 30 µL nuclease-free water.
RNA concentration was assessed by Qubit RNA High Sensitivity kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. UV absorbance at 230 nm, 260 nm, and 280 nm was measured using a nanodrop 2000 c spectrophotometer as an indicator of purity. RNA integrity and absence of genomic DNA contamination were confirmed using a TapeStation System (Agilent, Santa Clara, CA, USA) with the resulting gel images and electropherograms manually examined.
9
Quantitative PCR and Digital PCR Analysis
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Purified RNAs were treated with Turbo DNase (AM1907, Thermo Fisher Scientific) and quantified using the Qubit RNA high sensitivity kit (Q32852, Thermo Fisher Scientific). RNA integrity was analyzed using the Agilent RNA Pico 6000 kit on a 2100 Bioanalyzer instrument (Agilent Technologies). cDNA was synthesized using SuperScript III Reverse Transcriptase (18080044, Thermo Fisher Scientific), 100–500 ng of DNase-treated RNA and random hexamer primers. Quantitative PCR (qPCR) was performed on the ViiA 7 Real-Time PCR instrument (Thermo Fisher Scientific) using the PowerUp SYBR Green Master Mix (A25777, Thermo Fisher Scientific) and the respective primer sets (Supplemental Table 5 ). RT-negative controls were included in all qPCR reactions. Expression of target transcripts was quantified by ΔΔCt method using normalized Ct values. qPCR products were analyzed by 1.5% agarose gel electrophoresis and confirmed by Sanger sequencing. Droplet-based digital PCR (ddPCR) was performed on the QX100 Droplet Digital PCR system (Bio-Rad) using the QX200 ddPCR EvaGreen Supermix (1864033, Bio-Rad). PCR reactions were incubated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds and 58°C for 1 minute, with final incubations at 4°C for 5 minutes and at 90°C for 5 minutes. The average number of accepted droplets for the valid measurement results was approximately 17,000.
10
Labeling and Purification of RNA Oligonucleotides
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