Minigene splicing experiments were carried out in HEK293 cells transfected using lipofectamine 2000 (Invitrogen). RNA was extracted with Trizol (Invitrogen), and analysed using a one-step RT–PCR (PCR with reverse transcription) kit from Qiagen, both using the standard protocol. RT–PCR experiments used 100 ng of RNA in a 5-μl reaction using primers within the β-globin exons of pXJ41; PXJRTF (5′-GCTCCGGATCGATCCTGAGAACT-3′) and PXJB (5′-GCTGCAATAAACAAGTTCTGCT-3′). Reactions were analysed by agarose gel electrophoresis and quantified by capillary gel electrophoresis. For localization assays, HEK293 cells were fixed after 24 h using paraformaldehyde, mounted in VECTASHIELD with 4,6-diamidino-2-phenylindole (DAPI) and then directly visualized for green fluorescence protein (GFP) expression using fluorescence microscopy.
One step rt pcr pcr with reverse transcription kit
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The One-step RT–PCR (PCR with reverse transcription) kit is a laboratory instrument designed to perform reverse transcription and polymerase chain reaction (RT-PCR) in a single step. This kit enables the conversion of RNA into complementary DNA (cDNA) and subsequent amplification of target DNA sequences from the cDNA template.
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2 protocols using one step rt pcr pcr with reverse transcription kit
1
Minigene Splicing Assay in HEK293 Cells
2
Splicing Analysis of ETAA1 Exon 5 in HEK293 Cells
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A genomic region containing the weak 3ʹ splice site within ETAA1 exon 5 and flanking sequences were PCR amplified from human genomic DNA using the primers ETAA11295F
(5'-AAAAAAAAACAATTGGAACATGGAGCCAAACTAACTC-3') and ETAA11295R (5'-AAAAAAAAACAATTGTGATAGAATGGAGACTTGGGGA-3'), and cloned into pXJ41 (47) .
Splicing patterns were monitored after transfection into HEK293 cells with expression constructs encoding GFP, RBMX-GFP, RBMXL2-GFP, or Tra2β-GFP or deletion variants of the above plasmids as previously described (32, 50) . Splicing analysis was carried out in HEK293 cells after lipofectamine 2000 (Invitrogen) transfection of plasmids. RNA was extracted with TRIzol (Invitrogen), and analysed using a one-step RT-PCR (PCR with reverse transcription) kit from Qiagen, both using the standard protocol. RT-PCR experiments used 100 ng of RNA in a 5-μl reaction using a multiplex RT-PCR using primers:
5ʹ-GCTGGACATGTGGATTGGTG-3ʹ, 5ʹ-GTGGGAGCTGCATTTACAGATG-3ʹ and 5ʹ-GTGCTCCAAAAAGCCTCTGG-3 ʹ. Reactions were analysed and quantified by capillary gel electrophoresis.
(5'-AAAAAAAAACAATTGGAACATGGAGCCAAACTAACTC-3') and ETAA11295R (5'-AAAAAAAAACAATTGTGATAGAATGGAGACTTGGGGA-3'), and cloned into pXJ41 (47) .
Splicing patterns were monitored after transfection into HEK293 cells with expression constructs encoding GFP, RBMX-GFP, RBMXL2-GFP, or Tra2β-GFP or deletion variants of the above plasmids as previously described (32, 50) . Splicing analysis was carried out in HEK293 cells after lipofectamine 2000 (Invitrogen) transfection of plasmids. RNA was extracted with TRIzol (Invitrogen), and analysed using a one-step RT-PCR (PCR with reverse transcription) kit from Qiagen, both using the standard protocol. RT-PCR experiments used 100 ng of RNA in a 5-μl reaction using a multiplex RT-PCR using primers:
5ʹ-GCTGGACATGTGGATTGGTG-3ʹ, 5ʹ-GTGGGAGCTGCATTTACAGATG-3ʹ and 5ʹ-GTGCTCCAAAAAGCCTCTGG-3 ʹ. Reactions were analysed and quantified by capillary gel electrophoresis.
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