P-CAGE-25559 - P-CAGE-25559
RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, California, USA), including DNase treatment using the RNase-free DNase Set (Qiagen), according to the manufacturer’s instructions.
cDNA synthesis and purification
First-strand cDNA synthesis was accomplished by adding, to a total of 5 μg of total RNA, 1 μl of 0.5 μg/μl PAGE-purified T7dT primer (TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTNN), 1 μl of ten times diluted Lucidea Universal Scorecard control mixes (GE healthcare Bio-Sciences Corp., Piscataway, New Jersey, USA) and Rnase-free water to a final volume of 11 μl. After incubation for 10 min at 70ºC the mix was quickly chilled on ice and the contents were collected by quick centrifugation. A total of 4 μl 5X First Strand Buffer (Invitrogen, Carlsbad, CA), 2 μl 0.1M DTT (Invitrogen) and 1 μl of 10mM dNTP solution (Invitrogen) was added, and the contents were incubated at 37ºC for 2 min prior to addition of 2 μl SuperScript III reverse transcriptase enzyme (Invitrogen). The mix was incubated at 37ºC for two hours for reverse transcription and subsequently placed on ice.
Second strand cDNA synthesis was done by adding a total of 91 μl of Rnase-free water, 30 μl 5X Second Strand Buffer (Invitrogen), 3 μl 10 mM dNTPs, 4 μl E. coli Polymerase I (Invitrogen) and 1 μl RNase H (Invitrogen) to each first-strand synthesis reaction and incubating the mix for 2 hours at 16ºC. To stop the reaction, 10 μl of 0.5 M EDTA was added.
To purify the cDNA, each cDNA synthesis reaction mix was transferred into an phase-lock gel tube (Eppendorf, Hamburg, Germany) and 160 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed thoroughly. Phase-lock tubes were centrifuged for 5 min at 16060 × g. Following centrifugation, the supernatant was collected and placed in a fresh tube. An equal volume of 5 M NH4OAC, 1 μl of linear acrylamide and 2.5 volumes of 99.5% ethanol were added. After mixing, the tubes were put on ice for 10 min and then centrifuged for 30 min at 16060 × g at 4ºC. The supernatant was removed and washed twice with 500 μl of 80% ethanol with 5 min centrifugation at 16060× g after each wash. The purified cDNA was air-dried and re-suspended in 10 μl of RNase-free water.
In vitro transcription
In vitro transcription was performed using Ambion’s Megascript T7 kit (Ambion Inc., Austin, Texas, USA). A total of 5 μl purified cDNA was used as starting material, and the reaction was set up according to the manufacturer’s instructions. In vitro transcription reactions were incubated at 37ºC overnight. The RNA was purified on an Rneasy column (Qiagen, Valencia, California, USA) as follows. A total of 80μl of RNase-free water, 350μl of Buffer RLT (Qiagen) with β-mercaptoethanol and 250 μl of 99.5% ethanol was added to the RNA and the sample was mixed by pipetting. The sample was transferred to an RNeasy mini spin column and centrifuged for 15 s at 16060× g, transferred to a new collection tube and washed twice with 500 μl of Buffer RPE (Qiagen). The first wash was followed by a 15 s centrifugation at 8000 × g and the second wash was followed by a 2 min centrifugation at 16060 × g. After additional centrifugation for 1 min at 16060 × g the column was transferred to a 1.5 ml collection tube and purified RNA was eluted twice in 30 μl RNase-free water. The concentration of the in vitro transcribed RNA (aRNA) was checked on a Nanodrop (Wilmington, Delaware, USA) and the integrity was checked on a 1% agarose gel.
Amplification, Extracted product
|All experiments using protocol P-CAGE-25559: (E-ATMX-22, E-ATMX-23)|