Twenty-eight, 5- to 6-week-old, female, C3H/HeN mice were randomly assigned to groups. Each group of VEEV TC-83 infected mice (four mice per group) were humanely sacrificed at 1, 3, 5, 6, or 7 dpi. Mock-inoculated mice were sacrificed on 1 and 5 dpi. Following euthanasia, the whole brain, including the olfactory bulb, was removed from each mouse. Each brain was separated into eight portions: main olfactory bulb, piriform cortex, caudate-putamen, motor cortex, sensory cortex, thalamus, hippocampus, and cerebellum. Each section was homogenized in 1 mL of lysis buffer from the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher Scientific). Total RNA was isolated from each brain section using the MagMAX mirVana Total RNA Isolation Kit and the KingFisher Flex System (Thermo Fisher Scientific).
Magmax mirvana total rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Lithuania
The MagMAX mirVana Total RNA Isolation Kit is a product designed for the purification of total RNA, including small RNAs such as microRNAs, from a variety of sample types. The kit utilizes magnetic bead-based technology to capture and isolate the RNA, providing a reliable and efficient method for RNA extraction.
Automatically generated - may contain errors
Lab products found in correlation
Magna lyser instrument, by Roche (22 mentions)
Taqman rna to ct 1 step kit, by Thermo Fisher Scientific (21 mentions)
Kingfisher flex extraction robot, by Thermo Fisher Scientific (20 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Taqman advanced mirna cdna synthesis kit, by Thermo Fisher Scientific (7 mentions)
Kingfisher flex purification system, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Kingfisher flex system, by Thermo Fisher Scientific (3 mentions)
Rneasy minelute cleanup kit, by Qiagen (3 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Agilent 2100 bioanalyzer small rna chip, by Agilent Technologies (3 mentions)
112 protocols using magmax mirvana total rna isolation kit
1
VEEV TC-83 Infection in C3H/HeN Mice
2
RNA-Seq of Purified Rod Photoreceptors
3
Urine miRNA Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Urine single spot samples were collected at the day of biopsy and stored at −80 °C until miRNA extraction. MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania) with MagMAX magnetic bead technology was used to extract miRNA from urine samples according to the manufacturer’s protocol. The extracted miRNA was reverse transcribed into complementary DNA (cDNA) using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania).
Real-time quantitative polymerase chain reaction (qPCR) was performed to quantify miR-21 and microRNA-191-5p (miR-191) using TaqMan Advanced miRNA Assay and TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania). TaqMan microRNA assays IDs were 477975_mir (hsa-miR-21-5p) and 477952_mir (hsa-miR-191-5p).
MiR-191 was used for normalization as it is described as a stable gene showing a low degree of variation and high stability value [27 (link),28 (link)]. The relative gene expression levels were expressed by the ΔCt values (ΔCt = CtmiR-21 − CtmiR191, where Ct is the cycle threshold value) [29 (link)]. Lower ΔCt values indicate a higher relative expression of the miR-21. All experiments were carried out in duplicate and the arithmetic mean value was used for analysis.
Real-time quantitative polymerase chain reaction (qPCR) was performed to quantify miR-21 and microRNA-191-5p (miR-191) using TaqMan Advanced miRNA Assay and TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania). TaqMan microRNA assays IDs were 477975_mir (hsa-miR-21-5p) and 477952_mir (hsa-miR-191-5p).
MiR-191 was used for normalization as it is described as a stable gene showing a low degree of variation and high stability value [27 (link),28 (link)]. The relative gene expression levels were expressed by the ΔCt values (ΔCt = CtmiR-21 − CtmiR191, where Ct is the cycle threshold value) [29 (link)]. Lower ΔCt values indicate a higher relative expression of the miR-21. All experiments were carried out in duplicate and the arithmetic mean value was used for analysis.
4
RNA Extraction from Biological Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from the 50 mg of each sample using the MagMAX mirVana total RNA isolation kit with the MagMAX express 96 on program AM1830_DW (Applied Biosystems, Foster City, CA, USA). Following the manufacturer’s protocol, 302.1 µL of lysis binding mix (300 µL of lysis buffer and 2.1 µL of 2-Mercaptoethanol) was added to each sample, and the samples were vortexed for 15 s before being put into the 5× g for 5 min at 2000 rpm. The manufacturer’s protocol was modified slightly, thus 150 µL of the lysate was put into each well of the processing plate rather than 100 µL. To each sample on this plate, 20 µL of binding mix (10 µL RNA beads and 10 µL enhancer) was added, and the plate shook for 5 min using the plate shaker Lab-Line™ at 950 rpm.
In total, RNA was extracted from 116 samples, 9 blanks from the crushing stage and 2 negatives to check for contamination during the extraction process. RNA was quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and standardized to 50 ng/µL per sample using RNase free water before storage at −80 °C.
In total, RNA was extracted from 116 samples, 9 blanks from the crushing stage and 2 negatives to check for contamination during the extraction process. RNA was quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and standardized to 50 ng/µL per sample using RNase free water before storage at −80 °C.
5
Extracting RNA from Human Blood Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described previously 36 (link), total RNAs from serum, plasma and blood cells of human blood samples, cell lines and cell culture medium were extracted by using a magnetic beads-based assay (MagMAX™ mirVana™ Total RNA Isolation Kit, Applied Biosystems). An appropriate amount of synthesized cel-miR-39 (RuiBiotech) was added as external standard to each sample for estimation of extraction recovery. Reverse transcription reactions were conducted with specific miRNA/piRNA stem-loop primers using the Revert Aid First Strand cDNA Synthesis Kit (Thermo).
6
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression was measured by real-time quantitative PCR. Total quadriceps RNA was isolated using MagMAX mirVana Total RNA Isolation Kit (Applied BiosystemsTM, Waltham, CA, USA) with the Kingfisher Duo Prime instrument (Fisher Scientific, MA, USA) and was stored at −80 °C. Quantity and purity were measured with QubitTM RNA Broad Range (BR) and Integrity Quality (IQ) assay kits (InvitrogenTM, Carlsbad, CA, USA) using the Invitrogen Qubit 4 Fluorometer (InvitrogenTM, CA, USA). cDNA was synthesized from 1 μg of total quadriceps RNA with Maxima H Minus cDNA Synthesis Master Mix (Fisher Scientific, MA, USA). Real-time PCR was performed in triplicate in a total reaction volume of 15 μL using either PowerTrackTM SYBR Green Master Mix (Applied Biosystems, CA, USA) with primers described in Table 2 and measured in automated QuantStudio 3 Real-Time PCR System (Applied BiosystemsTM, CA, USA). Linear ranges and optimal RNA concentrations for each primer set were previously determined. Each primer sets were designed to span an exon/exon junction to minimize amplification of genomic DNA and obtained from Applied Biosystems. The acidic ribosomal phosphoprotein P0 (36B4) gene was used as housekeeping gene.
7
Profiling Human Plasma miRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used The TaqMan OpenArray Human MicroRNA Panels, QuantStudio™ 12K Flex (Applied Biosystems, Waltham, MA) for the initial screening step. The TaqMan OpenArray Human MicroRNA Panel is a fixed‐content panel (754 miRNAs) containing validated human TaqMan MicroRNA assays derived from Sanger miRBase release version 14.
EDTA‐plasma was prepared from whole blood samples at baseline and were stored at −80°C for further processing. Total RNA extraction was done from 100‐μL plasma samples using the MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems). Approximately 100 ng of total RNA was split and processed in parallel with Megaplex RT primer pools A and B and TaqMan MicroRNA Reverse Transcription Kit for preparing cDNA, followed by preamplification with TaqMan preamplification master mix with Megaplex preamplification primer pools A and B. The preamplified product was diluted and mixed in 1:1 ratio with TaqMan OpenArray Real‐Time PCR Master Mix and added onto the 384‐well OpenArray Sample Loading Plate. TaqMan OpenArray Human MicroRNA Panels were automatically loaded by the AccuFill System and then placed in the QuantStudio 12K Flex Real‐Time PCR System for polymerase chain reaction (PCR) cycling.
EDTA‐plasma was prepared from whole blood samples at baseline and were stored at −80°C for further processing. Total RNA extraction was done from 100‐μL plasma samples using the MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems). Approximately 100 ng of total RNA was split and processed in parallel with Megaplex RT primer pools A and B and TaqMan MicroRNA Reverse Transcription Kit for preparing cDNA, followed by preamplification with TaqMan preamplification master mix with Megaplex preamplification primer pools A and B. The preamplified product was diluted and mixed in 1:1 ratio with TaqMan OpenArray Real‐Time PCR Master Mix and added onto the 384‐well OpenArray Sample Loading Plate. TaqMan OpenArray Human MicroRNA Panels were automatically loaded by the AccuFill System and then placed in the QuantStudio 12K Flex Real‐Time PCR System for polymerase chain reaction (PCR) cycling.
8
RNA Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from cell lines and clinical tissues was extracted with Trizol reagent (Invitrogen), while total RNA from human serum samples was extracted with the MagMAX™ mirVana™ Total RNA Isolation Kit (Applied Biosystems). Reverse transcription reactions were conducted with oligo (dT) or specific miRNA/piRNA stem-loop RT primers using the Revert Aid First Strand cDNA Synthesis Kit (Thermo). Relative RNA levels determined by RT-qPCR were measured on the Light Cycler 480 II using the SYBR Green method. The primer sequences used are shown in Table S5 . For quantification of piRNAs, U6 small nuclear RNA was employed as an internal control in both cell lines and tissue samples. Serum samples were spiked in synthesized cel-miR-39 and cel-miR-54 (RuiBiotech) as exogenous controls. All experiments were performed in three biological replicates. The relative expression of RNAs was calculated as the power value (2-△△Ct).
9
Pluripotency and Differentiation Assessment of iPSC Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Applied Biosystems TaqMan®hPSC Scorecard™ Panel (Thermo Fisher Scientific) was used as an additional technique to assess pluripotency and tri-lineage differentiation potential of iPSC lines using real-time qPCR assays (Fig. 1 E, Supplementary Figs. 2–24E ). Total RNA from undifferentiated and EB differentiated iPSC lines was isolated using MagMAX™ mirVana™ Total RNA Isolation Kit (A27828), and 1 μg of RNA was used to make cDNA using the High Capacity cDNA Reverse Transcription Kit (4368813), both from Applied Biosystems. TaqMan qRT-PCR was carried out using the hPSC Scorecard 384w Fast plate (Life technologies, A15870) and QuantStudio 12 k Flex, following manufacturer protocol. We analysed the gene expression data from the TaqMan®hPSC Scorecard™ Panel using the web-based hPSC Scorecard™ Analysis Software (Thermo Fisher Scientific).
10
Quantification of Plasma miR375 Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from 100 µL plasma using the MagMax mirVana Total RNA Isolation Kit (Applied Biosystems, Foster City, California, USA). 10 ng of total RNA were reverse transcribed into cDNA using TaqMan™ MicroRNA Reverse Transcription Kit (4,366,596, Applied Biosystems). MiR375 levels were determined on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA) in duplicate qPCR reactions (3.35 ng cDNA/reaction) according to manufacturer’s instructions using the TaqMan™ MicroRNA Assay (#4,427,975, Assay ID 000564, Applied Biosystems) and the TaqMan™ Fast Advanced Master Mix (4,444,556, Applied Biosystems). Synthetic miR375 oligonucleotide (IDT, Iowa, USA) was used to determine cDNA synthesis efficiency and construct standard curves for absolute quantification during qPCR.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!