Whole RNA was extracted from human plasma using mirVana PARIS RNA Isolation Kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's liquid sample protocol. The RNA purity as well as the concentration extracted based on optical density at 260 and 280 nm. cDNA was synthesized from RNA via RT by utilizing gene-specific primers (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA) and Moloney Murine Leukemia Virus RT Kit, according to the following manufacturer's protocol, as follows: 42°C for 2 to 5 minutes, 42°C for 15 minutes, and 70°C for 50 minutes. The primer sequences for PCR amplification are summarized in Table 1 . qRT-PCR was utilized to assess candidate lncRNA expression levels with the StepOnePlus Real-Time PCR system. U6 was used as an internal control. The PCR conditions were as follows: 40 cycles at 95°C for 5 minutes, 95°C for 45 seconds, 55°C for 50 seconds, and 72°C for 15 seconds. The samples were analyzed in triplicate, and gene expression was quantified by normalizing expression of the target gene to the internal control utilizing the 2−ΔΔCq formula.[20 (link)]
Mirvana paris rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MirVANA PARIS RNA isolation kit is a product designed for the purification of total RNA, including small RNAs, from a variety of sample types. The kit utilizes a phenol-based extraction method to efficiently isolate high-quality RNA suitable for downstream applications such as RT-PCR, northern blotting, and microarray analysis.
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Lab products found in correlation
Rna 6000 nano chip, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Moloney murine leukemia virus rt kit, by Thermo Fisher Scientific (1 mentions)
Dnase, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Rnu6b, by Thermo Fisher Scientific (1 mentions)
Viia7 real time qpcr system, by Thermo Fisher Scientific (1 mentions)
Nanophotometry, by Implen (1 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Eukaryotic 18 s, by Thermo Fisher Scientific (1 mentions)
Taqman 2x universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
12 protocols using mirvana paris rna isolation kit
1
Plasma lncRNA Profiling by qRT-PCR
2
Quantitative RNA and Protein Analysis
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Total RNA from tumors was analyzed using mirVANA PARIS RNA isolation kit (Ambion, Austin, TX, USA) following manufacturer’s instructions. The transfection efficiency of mPEG-g-PEI in vitro was assessed by RT-PCR.
For protein analysis, the tumors were harvested on day 21 following different treatments as described earlier and washed with cold PBS. Proteins were extracted from C6 tumors using M-PER® protein extraction reagent (Pierce, Rockford, IL, USA). Twenty microgram of total protein extracted from cells was loaded per lane and separated by a 4%–20% SDS–Tris glycine polyacrylamide gel electrophoresis. Gels were blotted onto nitrocellulose membranes and membranes probed with SMAD5 and STAT6 antibodies.
For protein analysis, the tumors were harvested on day 21 following different treatments as described earlier and washed with cold PBS. Proteins were extracted from C6 tumors using M-PER® protein extraction reagent (Pierce, Rockford, IL, USA). Twenty microgram of total protein extracted from cells was loaded per lane and separated by a 4%–20% SDS–Tris glycine polyacrylamide gel electrophoresis. Gels were blotted onto nitrocellulose membranes and membranes probed with SMAD5 and STAT6 antibodies.
3
Whole Blood RNA Isolation Protocol
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Total RNA from whole blood
was isolated using the mirVANA PARIS RNA isolation kit (Ambion) following
the manufacturer’s instructions. Briefly, 400 μL of blood
lysate (200 μL whole blood + 200 μL RNA isolation buffer)
was combined with an equal volume of acid–phenol, vortexed,
and centrifuged at 13200g for 15 min. One half of
the aqueous phase was transferred to a fresh tube and vortexed with
1.25 × volume of 100% ethanol. Then, samples were applied to
glass fiber filter columns and centrifuged for 30 s followed by application
of washing buffers provided by the kit and according to the manufacturer’s
instructions. A volume of 50 μL of heated (95 °C), nuclease-free
water was used to elute the RNA from the column.
was isolated using the mirVANA PARIS RNA isolation kit (Ambion) following
the manufacturer’s instructions. Briefly, 400 μL of blood
lysate (200 μL whole blood + 200 μL RNA isolation buffer)
was combined with an equal volume of acid–phenol, vortexed,
and centrifuged at 13200g for 15 min. One half of
the aqueous phase was transferred to a fresh tube and vortexed with
1.25 × volume of 100% ethanol. Then, samples were applied to
glass fiber filter columns and centrifuged for 30 s followed by application
of washing buffers provided by the kit and according to the manufacturer’s
instructions. A volume of 50 μL of heated (95 °C), nuclease-free
water was used to elute the RNA from the column.
4
Plasma Small RNA Enrichment and Profiling
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Small RNA was enriched from all plasma samples using the mirVana PARIS RNA isolation kit (Ambion, Austin, TX). In brief, a 300 μL aliquot of plasma was thawed on ice and centrifuged at 14,000 rpm for 10 minutes to remove cells and cellular debris. Next, 250 μL of supernatant was lysed with an equal volume of 2x denaturing solution. For normalization of sample-to-sample variation during the RNA isolation procedures, 25 fmol of synthetic C. elegans miRNA (cel-miR-39, GE Dharmacon) was added to each denatured sample. Small RNAs were then enriched and purified following the manufacturer's protocol, with the exception that the enriched small RNAs were eluted in 20–30 μL of preheated nuclease-free water. DNase (Qiagen) treatment was used to remove any contaminating DNA. The RNA concentration was quantified using NanoDrop1000 (NanoDrop, Wilmington, DE) in all samples and ranged from 3 to 35 ng/μL.
5
miRNA Isolation and Profiling in Gastric Cancer
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miRNA was isolated using a mirVANA PARIS RNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions and the concentration was determined by 260/280 nm absorbance using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). miRNA microarray analysis performed by the KangChen Corporation (Beijing, China) was used to compare the miRNA expression profiles in one pair of an hTERT-positive gastric cancer tissue and an hTERT-negative corresponding adjacent tissues. Then, we used TargetScan to predict miRNAs with the potential to interact with hTERT.
6
Total RNA Extraction from Frozen Tissue
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Total RNA was extracted from previously-frozen tissue using the mirVana™ PARIS™ RNA isolation kit (Invitrogen; Cat #AM1556), according to the manufacturer’s instructions. The concentration and purity were determined by both spectrophotometry (A260, A260/280 respectively; NanoDrop 1000 Spectrophotometer; NanoDrop Technologies, Waltham, MA) and capillary electrophoresis (RNA Integrity Number [RIN], RNA 6000 Nano chip, Cat #5067-1511; Agilent Bioanalyzer 2100, Agilent Technologies).
7
Quantifying miRNA and GFP Expression in LAPC4 Cells
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Small RNA-containing total RNA was isolated from FACS sorted LAPC4 cells using the mirVana miRNA Isolation Kit (Invitrogen, cat# AM1561) or infected purified LAPC4 cells or tumors using the mirVana PARIS RNA Isolation Kit (Invitrogen, cat# AM1556) according to manufacturer’s instructions. miRNA was reverse transcribed to cDNA using TaqMan MicroRNa Reverse Transcription Kit (Applied Biosystems, cat# 4366596) according to manufacturer’s instructions. DNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, cat# ND-2000). qPCR was performed in technical triplicate using an MX3005P Real-time PCR System (Stratagene) with assays specific for each miRNA of interest as outlined in the Registered Report (Li et al., 2015 (link)). To detect copGFP expression, the following primers were used (Forward: 5’-AGGACAGCGTGATCTTCACC -3’; Reverse: 5’-CTTGAAGTGCATGTGGCTGT -3’) (Wang et al., 2008 (link)). PCR reactions were performed in technical triplicate with the following cycling conditions [1 cycle 95°C for 10 min – 40 cycles 95°C for 15 s, 60°C for 60 s] using an Mx3005P Real-time PCR System (Stratagene) and MxPro QPCR software (RRID:SCR_016375 ), version 4.1. Negative controls containing no cDNA template were included. Relative expression levels were determined using the ∆∆Ct method.
8
Total RNA Extraction from Tissue
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Total RNA was extracted from previously-frozen tissue using the mirVana PARIS RNA isolation kit (Invitrogen; Cat #AM1556), according to the manufacturer's instructions. The concentration and purity were determined by both spectrophotometry (A260, A260/280 respectively; NanoDrop 1000 Spectrophotometer; NanoDrop Technologies, Waltham, MA) and capillary electrophoresis (RNA Integrity Number [RIN] , RNA 6000 Nano chip, Cat #5067-1511; Agilent Bioanalyzer 2100, Agilent Technologies).
9
Validating RNA Sequencing Results via qPCR
10
Cerebrospinal Fluid Total RNA Extraction
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Total RNA was isolated using the mirVana PARIS RNA Isolation kit (Life Technologies), as described previously [18 (link)]. Briefly, 400 μl of CSF were diluted with equal volumes of 2x denaturing solution before equal volumes of acid-phenol:chloroform were added. Samples were centrifuged at 15,000 g for 10 min. The aqueous phase was mixed with 1.25 volumes of 100% ethanol. After sample application to a filter cartridge, washing was performed according to the manufacturer's instructions. RNA was eluted in 100 μl of RNAse-free water. RNA was quantified by nanophotometry (Implen).
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