Oligonucleotides were custom made by Integrated DNA Technologies, Inc. (Coralville, IA) and were used without purification. Non-DEPC treated DNase/RNase-free water was from Boston Bio Products (Ashland, MA). Bsm DNA polymerase, ultra-low range DNA ladder, RiboRuler High Range RNA ladder, Middlebrook 7H9 with OADC, Luria-Bertani (LB) broth, and Ambion Turbo DNA-free DNase kit were obtained from ThermoFisher Scientific (Waltham, MA). Hi-T7 RNA polymerase, Hi-T7 RNA polymerase buffer, ribonucleotide triphosphate (rNTP) mix, and deoxyribonucleotide (dNTP) mixes were purchased from New England BioLabs (Ipswich, MA, United States). GelRed® nucleic acid gel stain was from Biotium (Fremont, CA, United States). N-methylmesoporphyrin IX (NMM) and malachite green were purchased from Sigma-Aldrich (St. Louis, MO, United States). Dithiothreitol (DTT) and ammonium chloride were purchased from Acros Organics (Fair Lawn, NJ, United States). All other reagents were purchased from Fischer Scientific.
Ambion turbo dna free dnase kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Ambion TURBO DNA-free DNase kit is a laboratory tool designed to remove DNA from RNA samples. It utilizes a thermostable DNase enzyme to efficiently degrade DNA without affecting the integrity of the RNA.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotide, by Integrated DNA Technologies (2 mentions)
Gelred nucleic acid gel stain, by Biotium (2 mentions)
Non depc treated dnase rnase free water, by Boston BioProducts (2 mentions)
Middlebrook 7h9 with oadc, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Luria bertani broth, by Thermo Fisher Scientific (1 mentions)
Ammonium chloride, by Thermo Fisher Scientific (1 mentions)
Malachite green, by Merck Group (1 mentions)
Riboruler high range rna ladder, by Thermo Fisher Scientific (1 mentions)
Ultra low range dna ladder, by Thermo Fisher Scientific (1 mentions)
Hi t7 rna polymerase, by New England Biolabs (1 mentions)
Bsm dna polymerase, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Molecular imager gel doc xr, by Bio-Rad (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
C1000 touch thermocycler, by Bio-Rad (1 mentions)
Riboruler low range rna ladder, by Thermo Fisher Scientific (1 mentions)
Horizontal gel electrophoresis system, by Bio-Rad (1 mentions)
Seakem le agarose, by Lonza (1 mentions)
5 protocols using ambion turbo dna free dnase kit
1
In Vitro Transcription Protocol
2
NASBA Assay for Nucleic Acid Detection
3
Semi-quantitative RT-PCR for ERA Expression
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To analyze ERA expression, semi-quantitative RT-PCR was performed in duplicate on cDNA obtained from two different biological replicates of RNA from different tissues. Total RNA was extracted using the LiCl method43 (link). DNA contamination was removed using the Ambion TURBO DNA-free DNase kit according to the manufacturer’s instructions (Life Technologies). The RNA was reverse transcribed using the ImProm-II reverse transcription system (Promega) and the cDNA was used as template in semi-quantitative RT-PCR reactions. As a control, we simultaneously amplified an ACTIN fragment. Primers used for semi-quantitative RT-PCR are reported in Supplementary Table S1 .
4
RNA Isolation and Purification from Frozen Placenta Tissue
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A maximum amount of 20 – 25 mg frozen placenta tissues were distributed and homogenized in 300 µl of RNeasy lysis buffer (Qiagen, Valencia, CA, USA) using TissueLyser LT Adapter and stainless steel beads from Qiagen. RNA extraction was performed according to the manufacturerʼs instructions using RNeasy MiniKit (Qiagen, Valencia, CA, USA). Thereafter, Ambion TURBO DNA-
freeDNase kit (Life Technologies GmbH, Darmstadt, Germany) was used to remove contaminating DNA and to subsequently remove the DNase and divalent cations from extracted RNA samples. RNA purity and concentration were measured using Thermo Scientific NanoDrop 2000. In addition, further detecting of RNA integrity was done using Agilent RNA 6000 Nano Reagents Part I (Agilent Technologies, Waldbronn, Germany) and Bioanalyser Agilent 2100 from Agilent Technologies. Reverse transcribed complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster
City, CA, USA) as described by the manufacturer.
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A maximum amount of 20 – 25 mg frozen placenta tissues were distributed and homogenized in 300 µl of RNeasy lysis buffer (Qiagen, Valencia, CA, USA) using TissueLyser LT Adapter and stainless steel beads from Qiagen. RNA extraction was performed according to the manufacturerʼs instructions using RNeasy MiniKit (Qiagen, Valencia, CA, USA). Thereafter, Ambion TURBO DNA-
freeDNase kit (Life Technologies GmbH, Darmstadt, Germany) was used to remove contaminating DNA and to subsequently remove the DNase and divalent cations from extracted RNA samples. RNA purity and concentration were measured using Thermo Scientific NanoDrop 2000. In addition, further detecting of RNA integrity was done using Agilent RNA 6000 Nano Reagents Part I (Agilent Technologies, Waldbronn, Germany) and Bioanalyser Agilent 2100 from Agilent Technologies. Reverse transcribed complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster
City, CA, USA) as described by the manufacturer.
5
RNA Isolation and Quantitative RT-PCR
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For RNA isolation, cell pellets were thawed on ice, resuspended in 10 mM Tris pH 8 and lysed with 2 mg ml -1 lysozyme and 30 μg ml -1 lysostaphin, at 37 °C for 30 min. The lysates were transferred to Aurum RNA Binding Mini Columns and total RNA was extracted with Aurum™ Total RNA Mini Kit (Bio-Rad) following the manufacturer´s instructions. Contaminating DNA was removed by treatment with Ambion® TURBO DNA-free™DNase kit (Life Technologies). The concentration, purity and integrity of the RNA were evaluated in a Nanodrop ND-1000 UV-visible spectrophotometer (Thermo Fisher Scientific) and by gel electrophoresis.
For cDNA synthesis, 1 µg of RNA was reverse transcribed with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) using the Anchored-oligo (dT)18 and Random Hexamer primers. Quantitative real-time RT-PCR reactions were prepared with LightCycler® 480 SYBR Green I Master kit, using the primers listed in Table 4 and done in LightCycler® 480 (Roche). The fold change was calculated using the comparative CT method and 16S rRNA as reference gene. Assays were done for two independent biological samples analysed in triplicate.
For cDNA synthesis, 1 µg of RNA was reverse transcribed with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) using the Anchored-oligo (dT)18 and Random Hexamer primers. Quantitative real-time RT-PCR reactions were prepared with LightCycler® 480 SYBR Green I Master kit, using the primers listed in Table 4 and done in LightCycler® 480 (Roche). The fold change was calculated using the comparative CT method and 16S rRNA as reference gene. Assays were done for two independent biological samples analysed in triplicate.
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