Total RNA was isolated from frozen samples using miRNA isolation kits (Qiagen®, Germantown, MD, USA) according to the manufacturer’s protocol. Briefly, around 30 mg of snap-fresh tissue of HCC or adjacent non-tumorous liver were disrupted and homogenized. The lysate was then centrifuged and the supernatant was transferred to the gDNA Eliminator spin column. After centrifugation, the flow-through was transferred to the RNeasy spin column. RNA was extracted using the buffers RPE and RW1. The gDNA Eliminator spin columns, RNeasy spin column and buffers were all supplied in the Qiagen miRNA isolation kits. The concentration and quality of total RNA were measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) at 260 and 280 nm (A260/280) and confirmed by gel electrophoresis.
Mirna isolation kit
Manufactured by Qiagen
Sourced in Germany, United States
The MiRNA isolation kit is a laboratory tool designed to extract and purify microRNA (miRNA) from various biological samples. It provides a streamlined process for isolating miRNA, a class of small non-coding RNA molecules involved in gene expression regulation. The kit utilizes a silica-based membrane technology to efficiently capture and recover miRNA, enabling researchers to obtain purified miRNA samples for downstream analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex kit, by Takara Bio (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Cfx96 detection system, by Roche (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Mirna real time pcr assay kit, by Aidlab (2 mentions)
U6 snrna, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Qiagen (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Cfx96 detection system, by Bio-Rad (1 mentions)
Primescript rt master mix, by Toyobo (1 mentions)
Sybr pcr master mix, by Bio-Rad (1 mentions)
Sybr pcr master mix, by Roche (1 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rt2 mirna first strand kit, by Qiagen (1 mentions)
Rt pcr reverse transcription kit, by Qiagen (1 mentions)
34 protocols using mirna isolation kit
1
Total RNA Isolation from HCC Tissues
2
Quantification of miRNA-22 Expression
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Kidney samples were homogenized with Trizol. Total RNA was purified and miRNA was extracted using a miRNA Isolation Kit (Qiagen). 10 ng of RNA were reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). For cDNA synthesis, the reaction mixture was incubated at 16 °C for 30 minutes and then at 42 °C for 30 minutes; the enzyme was then inactivated at 85 °C for 5 min. 1.5 μL of the cDNA solution was then amplified using TaqMan Universal PCR Master Mix (Applied Biosystems), with miRNA-specific primer/probe (hsa-miR-22 000398, Thermo Fischer). The quantitative PCR was run on an ABI PRISM 7500 Real-time PCR system (Applied Biosystems) with an initial denaturation step at 95 °C for 10 minutes, followed by 40 cycles with a denaturation step at 95 °C for 15 seconds and an annealing/elongation step at 60 °C for 60 seconds. Each sample was run in triplicate for analysis. The miRNA expression levels were normalized to U6 snRNA (001973, Thermo Fischer).
3
Quantitative Gene Expression Analysis
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Total RNA was isolated using the miRNA isolation kit (Qiagen, cat #74106) according to the protocol of the manufacturers. Samples were treated with DNAse I (Qiagen cat# 79254). RNA was quantitated using Epoch/Take3 spectrophotometer system (BioTek Instruments, Inc.). Complementary DNA was generated with iScript cDNA Synthesis kit (Qiagen). We used StepOnePlus™ Real-Time PCR System to run a Taqman gene expression assay. Taqman expression assay probes for human genes have cross reactivity with corresponding rat genes expressed by H9c2 cells. To prepare the PCR reaction mix for each Taqman gene expression assay and samples on cycling plate: for one sample combine 10 µl of 2x Taqman fast universal PCR master mix, 1 µL Taqman gene expression assay, 5 µL water with 4 µL cDNA mix.
4
Quantifying Gene and miRNA Expression
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Total RNA was extracted from periodontal tissue and cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated with PrimeScript RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). The expression level of genes was measured by qPCR in a Bio-Rad CFX96™ Detection System (Roche, Sweden) with SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). Small RNA was extracted from cells with an miRNA isolation kit (Qiagen, Hilden, Germany), and cDNA was generated with an miRNA reverse transcription kit (Shenggong, Shanghai, China). The expression level of miRNAs was measured by qPCR in a Bio-Rad CFX96™ Detection System with SYBR PCR Master Mix. U6 was used as the internal reference. The primers used are shown in Supplementary Table S1 .
5
RNA Isolation from Periodontal Ligament Cells
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RNA was isolated from the PDLCs with or without LPS‐treated with the miRNA Isolation Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. The purity and quantity of RNA were measured by NanoDrop (ND‐1000 spectrophotometer; Thermo Scientific, Wilmington, DE, USA). The samples were used immediately or stored at −80°C.
6
Profiling miRNA Expression in IL-6-Treated INS-1 Cells
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Total RNA from INS-1 cells treated with 20 ng/mL IL-6 was extracted using the Qiagen miRNA isolation kit. Extracted miRNA was reverse transcribed to single-strand cDNA with RT2 miRNA first strand kit (Qiagen). Real-time quantitative PCR analysis (Rat miRNome RT2 miRNA PCR array, Qiagen) was performed using SYBR master mix using the ABI 7900 Real-time PCR system according to the protocols provided by the manufacturer (Applied Biosystems). Primer sequence was as follows: miR-101a (rno miR-101a miScript Primer assay, Qiagen cat# MS00012950), miR-122a (rno miR-122a miScript Primer assay, Qiagen cat# MS00000315), and miR-181c (rno miR-181c miScript Primer assay, Qiagen cat# MS MS00013132). The relative mRNA transcript levels were calculated according to 2Δ–CT method, in which ΔCT represents the difference in threshold cycle values between the target miRNA and Rnu6, which was used as an internal control.
7
Apoptosis Induction in RL95-2 Cervical Cancer Cells
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Human cervical cancer cell line RL95-2 was provided by the Department of Pathophysiology, Anhui Medical University. RT-PCR reverse transcription kit and miRNA isolation kit were purchased from Qiagen, Inc. (Valencia, CA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Tetramethyl azoline blue (MTT) and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA). Lipofectamine™ 2000 transfection reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Annexin V-FITC/PI apoptosis kit was purchased from BestBio (Shanghai, China). The study was approved by the Ethics Committee of Chongming Branch Hospital, Affiliated Xinhua Hospital, School of Medicine, Shanghai Jiaotong University (Suizhou, China). Signed informed consents were obtained from the patients or the guardians.
8
Validating miRNA Expression in Progressive MS
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Expression levels of miRNAs were conducted in a separate validation cohort consisting of six progressive MS brain samples (Table 1 , MS7‐12). To evaluate and validate miRNA expression, small‐sized RNA was isolated from demyelinated lesions (WML) and surrounding NAWM with a Qiagen miRNA isolation kit as per manufacturer's instructions. Isolated miRNAs were reverse‐transcribed to cDNA with a TaqMan miRNA RT Kit (Applied Biosystems, #4366596) as recommended by the supplier. Each sample was run in triplicate. The expression of selected miRNAs (Table S1 ) was validated using TaqMan miRNA assays (cat# 4427975) with PCR efficiency of 100% (±10%). RNU43 (assay ID #001095) and U6 snRNA (assay ID #001973) were used as endogenous controls in the reaction. To rule out inter‐assay variation, endogenous controls were also profiled in each plate separately. Delta Ct values were used to determine relative expression changes (fold change, 2−ΔΔCT). Groups in RT‐PCR analysis were compared using Student's t‐tests and a P‐value <0.05 was considered as significant. All quantitative data are expressed as mean ± standard error of the mean (±sem).
9
Quantitative PCR Protocol for Gene Expression
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Total RNA was isolated from tissue samples using the miRNA isolation kit (Qiagen, Germantown, MD) according to the protocol provided by the manufacturer. The cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using gene-specific primers (Supplemental Table S3 ) and the Roche SYBR green master mix. Samples were run on a LightCycler 480 II system (Roche, Pleasanton, CA) and analyzed using the Roche LightCycler 1.5.0 software26 (link). All qPCR targets were quantified based on standard curves ran on the sample plate. The mRNA levels of specific genes were normalized to the mRNA levels of housekeeping genes of the same sample. The mRNA levels of housekeeping genes eukaryotic translation initiation factor 2A (Eif2a, Fig. 2 a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Fig. 2 d,g), ribosomal protein L13a (Rpl13a, Fig. 3 d), and β2 microglobulin (B2M, Supplemental Fig. S2 ) were used for normalization.
10
Validating DEmiRNAs in Coronary Artery Disease
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qPCR was conducted using 12 new samples of EAT (n = 6 CAD and n = 6 non-CAD) to validate select DEmiRNAs (hsa-146b-5p, hsa-26b-5p, hsa-21-5p, hsa-320a) in the EAT of patients with CAD. miRNAs were isolated from the EAT of all patients using a commercially available miRNA isolation kit (QIAGEN, Germantown, MD, USA). Reverse transcription of the miRNAs was conducted using the miRCURY LNA RT Kit (QIAGEN, Germantown, MD, USA). PCR primers for hsa-miR-146b-5p, hsa-miR-26b-5P, hsa-miR-21-5p, and hsa-miR-320a were obtained from Qiagen. qPCR was conducted on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) using the cycle settings recommended in the miRCURY LNA RT Kit (QIAGEN, Germantown, MD, USA). ΔCt values were determined for each miRNA in each sample using the spliceosomal RNA U6 for normalization. Unpaired t-test (using ΔCt values) was used to determine statistically significant differential expression of the miRNAs between patients with and without CAD.
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