Pulverized uterus tissue was incubated for five minutes in trizol reagent. After centrifugation at 10.000×g for 10 min at 4°C, the supernatant was transferred to a Maxtract tube (Qiagen, Copenhagen, Denmark), and chloroform and RNase free water was added. Following centrifugation at 12.000×g for 10 min at room temperature the colorless water phase was transferred to a clean tube and added isopropanol. After 10 min incubation the mix was transferred to a spin column and DNase treated using SV total RNA purification kit (Promega, Nacka, Sweden). The following RNA wash was performed with SV total RNA purification kit (Promega, Nacka, Sweden) according to the guidelines from the manufacturer. Total RNA was eluted in H2O. Concentration and purity of the RNA was measured by spectrophotometry on a nanodrop and bio-analyzer. RIN values above 7 were accepted for further analysis. The RNA was kept at −80°C until further use.
Sv total rna purification kit
Manufactured by Promega
Sourced in Sweden, United States
The SV Total RNA Purification Kit is a laboratory equipment product designed for the isolation and purification of total RNA from various sample types, such as cells, tissues, or blood. The kit utilizes a silica-membrane-based technology to capture and purify RNA without the use of organic solvents. The purified RNA can be used in downstream applications such as reverse transcription and real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dig pcr nucleotide mix, by Roche (2 mentions)
Dig luminescent detection kit, by Roche (2 mentions)
Quantifast sybr green pcr kit, by Qiagen (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rifampicin, by Serva Electrophoresis (1 mentions)
Positively charged membrane, by Roche (1 mentions)
Maxtract tube, by Qiagen (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Dnase 1, by Promega (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
10 protocols using sv total rna purification kit
1
Uterus Tissue RNA Extraction
2
Transcriptional Regulation Analysis
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Bacteria grown under the required growth conditions were pelleted and RNA was isolated using the SV total RNA purification kit (Promega) as described [34 (link)]. Total RNA (20 μg) was separated on MOPS agarose gels (1.2%), transferred by vacuum blotting for 1.5 h onto positively charged membranes (Whatman) in 10 x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate, pH7) using a semi-dry blotting system and UV cross-linked. Prehybridization, hybridization to DIG-labelled probes and membrane washing were conducted using the DIG Luminescent Detection Kit (Roche, Germany) according to the manufacturer's instructions. The DIG-labelled PCR fragments used as probes were produced by PCR using the DIG-PCR nucleotide mix (Roche, Germany) as described [34 (link)] with the following primer pairs: for the lcrF transcript—I214/I303, for the csrB and csrC transcripts—555/556 and 583/I82, for the rne transcript—IV529/IV530 and the pnp transcript—IV527/IV528 (see S2 Table ).
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.
3
RNA Extraction and Northern Blot Analysis
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Overnight cultures were grown to stationary phase (OD600 of 3). 2.5 ml culture were withdrawn, mixed with 0.2 volume of stop solution (5% water-saturated phenol, 95% ethanol) and snap-frozen in liquid nitrogen. After thawing on ice, bacteria were pelleted by centrifugation (2 min, 14.000 rpm, 4°C), and RNA was isolated using the SV total RNA purification kit (Promega) as described by the manufacturer. RNA concentration and quality were determined by measurement of A260 and A280. Total cellular RNA (10 μg) was mixed with loading buffer (0.03% bromophenol blue, 4 mM EDTA, 0.1 mg/ml EtBr, 2.7% formaldehyde, 31% formamide, 20% glycerol in 4 × MOPS buffer) and was separated on agarose gels (1.2%), transferred overnight onto positively charged membranes (Roche) in 20 × SSC and UV cross-linked. Prehybridization, hybridization to DIG-labeled DNA probes and membrane washing were conducted using the DIG luminescent Detection kit (Roche) according to the manufacturer’s instructions. The csrC and csrB transcripts were detected with a DIG-labeled PCR fragment (DIG-PCR nucleotide mix, Roche) with primer pair 23/24 and 25/26 (Supplementary Table 2 ), respectively.
4
16S rRNA Gene Amplification from cDNA
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Total RNA of pMF1-harboring M. fulvus 124B02 was extracted using an SV Total RNA Purification Kit (Promega USA). Residual genomic DNA (gDNA) was removed and cDNA was synthesized using a Prime ScriptTM RT Reagent Kit with gDNA Eraser (Takara, China).The 16S rRNA gene sequence was amplified from the cDNA template to evaluate the quality of cDNA sample. The cDNA products were further amplified using specific primer pairs to determinate the co-transcription.
5
Quantitative PCR Analysis of Melanocytes
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Total RNA was extracted from melanocytes with SV total RNA purification kit (Promega, Shanghai, China). Reverse transcript reaction was performed using QuantiTect Reverse Transcription Kit (Qiagen, Germany). Real time PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen). The expression levels of each gene was normalized against β-actin using the comparative Ct method, and expressed as percentage of control, with the control as 1. Sequences of primers are listed on the Table S1 .
6
Gene Expression Analysis of Fungal Mycelia
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Mycelia harvested at specific time points were powderized using in N2 (l) and total RNA was extracted from the frozen pellet using the SV total RNA purification Kit (Promega). RNA samples were subjected to Dnase I (Promega) digestion according to the manufacturer instructions. First-strand cDNA synthesis was accomplished using Invitrogen’s SuperScript™ Kit and second step PCR was carried out under previously indicated conditions [1 (link)]. Control RT-PCR was similarly performed in the absence of reverse transcription to check for DNA contamination after Dnase I digestion required during RNA preparation. The qPCR was performed using MaximaTM SYBR Green qPCR Mix (MBI) and Applied Biosystem’s 7500 Fast Real-time PCR system. 16S rDNA was used as the internal control. All of the primers used are shown in Additional file 1 : Tables S4 and S5. All qPCR assays were repeated in triplicate. Statistical analysis was carried out using SPSS version 13. One-vay ANOVA at a 95% confidence level (p < 0.05 and p < 0.01) was used to evaluate the significance of the difference between each sample.
7
RNA Extraction from M9 Bacterial Cultures
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LB overnight cultures were washed with water and diluted to a final OD600 of 0.05 in 250 ml flasks containing 25 ml of modified M9 medium without iron addition [14 (link)]. The cultures were incubated at 37°C in a shaking incubator until the exponential phase of growth (OD600 = 0.2). Synthesis and degradation of RNA were blocked by adding 1/5 volume of stop-solution (90% ethanol/10% phenol) [15 (link)]. The RNA was purified using the Promega SV total RNA purification kit according to the manufacturer’s instructions. Quantification of the RNA was performed by measuring the 260 nm absorbance on a Nanodrop 1000 spectrophotometer.
8
Quantifying MMP and TIMP Genes
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After the treatments, cells were harvested, and total RNA was extracted from different treated cells as described earlier with SV total RNA purification kit (Promega Corporation, Fitchburg, WI, USA). Reverse transcript reaction was performed using QuantiTect Reverse Transcription Kit (Qiagen NV, Venlo, the Netherlands). Real-time polymerase chain reaction was performed using QuantiFast SYBR Green PCR Kit (Qiagen NV). The expression levels of each gene were normalized against β-actin using the comparative Ct method and expressed as percentage of control, with the control as 1. Sequences of primers were MMP-2 forward (5′-ACCGAGGACTATGACCGGGATAA-3′), reverse (5′-GCTCTCATACTTGTTGCCCAGGA-3′); MMP-9 forward (5′-GCCCTGGAACTCACACGACA-3′), reverse (5′-TTGGAAACTCACACGCCAGAAG-3′); TIMP-1 forward (5′-ATGCGGCCGCATGATGGCCCCCTTTGCATC-3′), reverse (5′-ATCCCGGGTCATCGGGCCCCAAGGGATC-3′); and TIMP-2 forward (5′-GTAGTGATCAGGGCCAAAG-3′), reverse (5′-TTCTCTGTGACCCAGTCCAT-3′).
9
Steady-State and Stability Analysis of CsrB and CsrC RNAs
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Primer extension analysis was performed to determine the steady-state level and the stability of the CsrB and CsrC RNA from strains YPIII and YP80 (YPIII Δhfq). At an OD600 of 2.0 (early stationary phase), rifampicin was added to a final concentration of 500 μg ml–, after 0, 10, 20, 30, and 60 min, 2 ml aliquots were withdrawn and total RNA was extracted of the samples using the SV total RNA purification kit (Promega) as described by the manufacturer. Annealing was performed with 5 μg extracted RNA and the 5′-Dig-labeled oligonucleotides (primer 5′-CTGAAGACACATCTTCC-3′ for CsrB, primer 5′- CCTGAGTAACTGTGCTCC-3 for CsrC, and primer 5′-CCCACACTACCATCGGCGC-3′ for 5S RNA) in 20 μl of 1x First Strand Buffer (Invitrogen) by slow cooling of the sample (0.01°C/sec) including 8 mM dNTPs with 200 U Superscript II reverse transcriptase (Invitrogen) was added and incubated for 1 h at 42°C. The size of the Dig-labeled reaction products was determined on a denaturing 4% DNA sequencing gel by a detection procedure as described (Heroven et al., 2008 (link)).
10
Quantitative RT-PCR Analysis of camE
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Total RNA of each mutant was extracted using the SV total RNA purification Kit (Promega) and digested by DNase I (Takara). First-strand cDNA synthesis was accomplished using Invitrogen’s SuperScript™ Kit, and second step PCR was carried out under the following conditions: 94 °C for 5 min, 25 cycles of denaturation (94 °C for 25 s), annealing (60 °C for 25 s), and extension (72 °C for 45 s), and a single extension at 72 °C for 10 min. A negative control was accordingly performed in the absence of template to check for DNA contamination after the DNase I digestion required for RNA purification.
Quantitative real-time reverse transcription PCR (qPCR) was performed using the Maxima™ SYBR Green qPCR Mix (MBI) and Applied Biosystem’s 7500 Fast Real-time PCR system. 16S rDNA was used as the internal control. The primers used to analyze the camE gene (NCBI accession number: WP_103055331.1) and 16S rDNA are shown in and Additional file1 : Table S17.
Quantitative real-time reverse transcription PCR (qPCR) was performed using the Maxima™ SYBR Green qPCR Mix (MBI) and Applied Biosystem’s 7500 Fast Real-time PCR system. 16S rDNA was used as the internal control. The primers used to analyze the camE gene (NCBI accession number: WP_103055331.1) and 16S rDNA are shown in and Additional file
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