Total RNA was isolated from resected frozen specimens by using RNAspin Mini (GE Healthcare, Tokyo, Japan) and the first-strand cDNA was synthesized from 1 μg RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA), according to the manufacturer’s protocol. Real-time quantitative PCR analysis was carried out using an ABI Prism 7700 sequence detector system (Applied Biosystems). All primer/probe sets were purchased from Applied Biosystems. PCR was carried out using the TaqMan Universal PCR Master Mix (Applied Biosystems) using 1 μl of cDNA in a 20 μl final reaction volume. The PCR thermal cycle conditions were as follows: initial step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 min. The expression level of the housekeeping gene β2-microglobulin was measured as an internal reference with a standard curve to determine the integrity of template RNA for all specimens. The ratio of the mRNA level of each gene was calculated as follows: (absolute copy number of each gene)/(absolute copy number of β2-microglobulin).
Rnaspin mini
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
The RNAspin Mini is a compact and efficient RNA extraction kit designed for small-scale RNA purification from a variety of sample types. The kit utilizes a simple silica-based membrane technology to isolate high-quality total RNA, suitable for downstream applications such as RT-PCR, Northern blotting, and library preparation.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Miscript pcr system, by Qiagen (3 mentions)
Miscript sybr green pcr kit, by Qiagen (3 mentions)
Miscript 2 rt kit, by Qiagen (3 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (2 mentions)
Abi prism 7700 sequence, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (1 mentions)
High capacity cdna reverse transcription kit, by GE Healthcare (1 mentions)
Taqman fast universal pcr master mix 2x, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Megaplex rt primers human pool a, by Thermo Fisher Scientific (1 mentions)
Taqman human microrna array a, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
13 protocols using rnaspin mini
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
RNA Extraction and Gene Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extraction for RNA-seq was done from 1 × 106 CD138-selected cells from patient number 3 using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Similarly, for the INA-6 cell line microarray the RNA was extracted from the cells using RNASpin mini (GE Healthcare) after 72h of treatment with 2 μM UNC1999. The labeling of the RNA for microarray and hybridization were done according the Affymetrix manufacturer's protocol (Affymetrix). The data was analyzed using GeneSpring 13 software using quantile normalization and ExonRMA 16 was used for summarization. The differentially regulated genes were obtained using moderated t-test with the p < 0.02 and fold change 1.5. cDNA preparations were done using Invitrogen kit as per the manufacturer's protocol. For qPCR TaqMan® (Invitrogen) and SYBR® Green (Invitrogen) chemistry was used according the manufacturers’ protocols (Table S4 ).
3
Quantifying Corneal Epithelial Marker Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate marker gene expression of corneal epithelial cells, total RNA from stratified cells cultured on the DCL was extracted using RNAspin Mini according to the manufacturer's protocol (GE Healthcare Bio-Sciences AB, Little Chalfont, UK). Equal amounts (1 μg) of total RNA were reverse transcribed into cDNA by using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The transcribed complementary DNAs were used for real-time polymerase chain reaction (RT-PCR). The PCR primers and annealing temperatures for target genes were designed based on published human gene sequences (Table 3 ). The amplified products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining, and GAPDH was used as an internal loading control. Images were captured on an ImageQuant LAS 4000 (GE Healthcare).
4
Quantification of macrophage phenotype markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from infected BMDMs was isolated using RNAspin Mini according to the manufacturer’s instructions (GE Healthcare) and reverse-transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad). Gene expression was determined by amplification with specific primers and quantification by SybrGreen (Life). The relative gene expression was calculated by the 2−ΔCt method. For qPCR, a thermocycler ABI Prim 7500 (Applied Biosystems, Foster City, CA) was used. Primer sequences used to determine macrophage phenotype were: Arg1 (forward, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′; reverse, 5′-AGGAGCTGTCATTAGGGACATC-3′), Mrc1 (CD206) (forward, 5′-CCATTTATCATTCCCTCAGCAAGC-3′; reverse, 5′-AAATGTCACTGGGGTTCCATCACT-3′), Fizz-1 (forward, 5′-ACTGCCTGTGCTTACTCGTTGACT-3′; reverse, 5′-AAAGCTGGGTTCTCCACCTCTTCA-3′), Cd86 (forward, 5′-CARGGGCTTGGCAATCCTTA-3′; reverse, 5′-AAATGGGCACGGCAGATATG-3′), Nos2 (iNOS) (forward, 5′-CAGCTGGGCTGTACAAACCTT-3′; reverse, 5′-GCTCTGTTGAGGTCTAAAGGCT-3′), Gapdh (forward, 5′-AACTTTGGCATTGTGGAAGG-3′; reverse, 5′-ACACATTGGGGGTAGGAACA-3′), and Il-6 (forward, 5′-TTCCATCCAGTTGCCTTCTTG-3′; reverse, 5′-AGGTCTGTTGGGAGTGGTATC-3′).
5
Quantitative Analysis of Antioxidant Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GCL is the rate-limiting enzyme in de novo GSH synthesis and is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits. We examined the effect of EPS on GCLM, which is limiting in most cell types and tissues [23] (link). Quantitative RT-PCR analysis was used to measure mRNA levels. Total RNA from treated cells was extracted with RNAspin Mini (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's protocol. mRNAs were reverse-transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems). Primers for bovine GCLM (Bt03232353-m1), bovine Nrf2 (Bt03251879-m1), bovine HO-1 (Bt03218621-m1), and bovine Trx-1 (Bt03222877-g1) were purchased from Applied Biosystems. mRNA levels were acquired from the value of the threshold cycle (Ct) of GCLM, Nrf2, HO-1, or Trx-1 normalized to that of GAPDH. Relative mRNA levels were compared and expressed as percentage of control levels. Data are representative of three experiments.
6
Gene Expression Analysis by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were extracted for total RNA using the RNAspin Mini (GE Healthcare UK Ltd, Buckinghamshire, UK) following manufacturer’s instructions. The total RNA was reverse-transcribed into cDNA using the Tetro cDNA Synthesis Kit (Bioline, MA, USA). The cDNA was amplified for 35 cycles using specific primers (S1 Table ). Quantitative data were calculated using the 2-ΔΔCT method and normalized to GAPDH expression.
7
Quantifying RBX1 mRNA Levels in EC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from EC tissues, non-cancer tissues and human EC cell lines using RNAspin Mini (GE Healthcare), and the first-strand cDNA was synthesized from 1 µg RNA using the high-capacity cDNA reverse transcription kit (cat. no. 4368813; Applied Biosystems; Thermo Fisher Scientific, Inc.) as previously described (16 (link),23 (link),24 (link)). For RT-qPCR, cDNA was amplified using TaqMan Fast Universal PCR Master Mix (2X; Applied Biosystems; Thermo Fisher Scientific, Inc.) with gene-specific primers and a lysate probe on a StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The thermal cycling conditions were 95°C for 20 sec, followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. qPCR experiments for each gene were carried out on 3 separate occasions. All primer/probe sets were purchased from Applied Biosystems; Thermo Fisher Scientific, Inc. (RBX1; Hs00360274m1, β2-microglobulin; Hs00984230_m1). Expression levels of β2-microglobulin, a housekeeping gene, were measured as an internal reference with a standard curve to determine the integrity of template RNA for all the specimens. The ratio of the RBX1 mRNA levels were calculated as follows: (Absolute copy number of RBX1)/(absolute copy number of β2-microglobulin) (25 (link)).
8
Quantification of miR-21 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of miR-21 was tested by miScript PCR System (QIAGEN, Hilden, Germany). Total cellular RNA and miRNAs were isolated from HUVECs incubated or not with exosomes using the RNAspin Mini (GE Healthcare Science, Uppsala, Sweden). Reverse transcription reactions were performed using miScript II RT Kit (QIAGEN, Hilden, Germany) as described by the manufacturer's instructions. We used miScriptHiSpec Buffer for cDNA synthesis to detect mature miRNA and miScriptHiFlex Buffer for cDNA synthesis to enable the quantification of precursor miRNA. Quantitative Real Time PCR was performed using miScript SYBR Green PCR Kit (QIAGEN, Hilden, Germany). Mature miR-21 was detected by miScript Primer Assay and precursor miR-21 by miScript Precursor Assays according to manufacturer's instructions. RNU6-2 was used as endogenous control. Expression levels of miRNAs and pre-miRNA were determined using the comparative Ct method to evaluate changes in Ct and ultimately fold and percent change. An average Ct value for each RNA was obtained from triplicate reactions.
9
Profiling miRNA Expression in LAMA84 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA and miRNAs were isolated from LAMA84 cells and exosomes using the RNAspin Mini (GE Healthcare Science, Uppsala, Sweden) and analysed through 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 600 ng of total RNA was reverse transcribed using Megaplex™ RT Primers Human Pool A (Life Technologies, Carlsbad, California, U.S.) according to manufacturer’s instructions. Conditions for the reverse transcription reaction were as follows: 16°C for 2 minutes, 42°C for 1 minute, 50°C for 1 second for 40 cycles, 85°C for 5 minutes then hold at 4°C. Obtained cDNA was diluted, mixed with TaqMan Gene Expression Master Mix and loaded into each of the eight fill ports on the TaqMan® Human MicroRNA Array A (Life Technologies, Carlsbad, California, U.S.). The array was centrifuged at 1200 rpm twice for 1 minute each and then run on ABI-PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the manufacturer’s recommended program. Data were quantified using the SDS 2.1 software and normalized using the miR-18b as endogenous control or RNU6-2. The cycle threshold (Ct) value, which was calculated relatively to the endogenous control, was used for our analysis (ΔCt). The 2-ΔΔCT (delta-delta-Ct algorithm) method was used to calculate the relative changes in gene expression.
10
Quantitative Analysis of Key Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA was isolated from K562 and LAMA84 cells and exosomes using the RNAspin Mini (GE Healthcare Science, Uppsala, Sweden). For PTEN, VEGF and BCR-ABL mRNA detection, 1 μg of total RNA were reverse transcribed using the High Capacity cDNA Archive kit (Life Technologies, Carlsbad, California, U.S.), according to manufacturer's instructions. PTEN, VEGF and BCR-ABL transcript levels were measured by TaqMan Real Time PCR, using TaqMan gene expression assay for PTEN (Hs00262123 m1), VEGF (Hs0090005.m1) and VEGF (Hs0090005.m1) (Life Technologies, Carlsbad, California, USA). Data were analyzed as previously described. Changes in the target mRNA content relative to GAPDH were determined using the comparative Ct method as described in the previous paragraph.
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!