umrt reverse transcription kit  (RNAConnect Inc)

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with iScript, SuperScript III (SSIII), and uMRT. (B) uMRT performs rolling circle reverse transcription at lower temperatures. 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with uMRT at room temperature (25 °C), 30 °C, and 42 °C. (C) Sanger sequencing results of concatemeric RT-PCR products by SuperScript III and uMRT. The circRNA sequence (top, green annotation) shows the circRNA junction (annotated with the orange triangle).

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) SuperScript III and uMRT were compared in their ability to reverse-transcribe larger circRNAs. The following circRNAs were used as input for the reverse transcription: lncATV (420 nt), circ-Znf609 (935 nt), and CVB3-EGFP (1596 nt). (B) Nanopore sequencing results of RT-PCR amplicons. Sequencing results across the junctions are shown (yellow highlighted region). Note that circ-lncATV and circ-Znf609 have identical splicing junctions due to the synthesis method. The whole sequence alignment is shown by the grey bar.

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription, Nanopore Sequencing, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) RT-PCR of circHIPK3 using uMRT, SuperScript III (SSIII), and SuperScript IV (SSIV). The expected RT-PCR product for circHIPK3 is 1,099 nt. (B) Method overview for detecting natural circRNAs. RNA extraction from HEK293T cells is treated with RNase R to remove linear RNA. (C) Nanopore sequencing results of the uMRT RT-PCR amplicon. Sequencing results across the junction are shown (yellow highlighted region). The whole sequence alignment is shown by the grey bars.

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Nanopore Sequencing, Amplification, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with iScript, SuperScript III (SSIII), and uMRT. (B) uMRT performs rolling circle reverse transcription at lower temperatures. 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with uMRT at room temperature (25 °C), 30 °C, and 42 °C. (C) Sanger sequencing results of concatemeric RT-PCR products by SuperScript III and uMRT. The circRNA sequence (top, green annotation) shows the circRNA junction (annotated with the orange triangle).

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) SuperScript III and uMRT were compared in their ability to reverse-transcribe larger circRNAs. The following circRNAs were used as input for the reverse transcription: lncATV (420 nt), circ-Znf609 (935 nt), and CVB3-EGFP (1596 nt). (B) Nanopore sequencing results of RT-PCR amplicons. Sequencing results across the junctions are shown (yellow highlighted region). Note that circ-lncATV and circ-Znf609 have identical splicing junctions due to the synthesis method. The whole sequence alignment is shown by the grey bar.

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription, Nanopore Sequencing, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) RT-PCR of circHIPK3 using uMRT, SuperScript III (SSIII), and SuperScript IV (SSIV). The expected RT-PCR product for circHIPK3 is 1,099 nt. (B) Method overview for detecting natural circRNAs. RNA extraction from HEK293T cells is treated with RNase R to remove linear RNA. (C) Nanopore sequencing results of the uMRT RT-PCR amplicon. Sequencing results across the junction are shown (yellow highlighted region). The whole sequence alignment is shown by the grey bars.

Article Snippet: Add 1 μL of RNase R to 20 μg of RNA in a 20 μL reaction volume. b. Incubate at 37 °C for 30 min. c. Remove the RNase R enzyme using a Zymo RNA Clean and Concentrator-25 column and elute the remaining RNA using 25–50 μL of nuclease-free water. d. The eluted RNA can be used as a template for section C . C. uMRT reverse transcription The uMRT reverse transcription was adapted from the manufacturer’s protocol (RNAConnect, R1002S).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Nanopore Sequencing, Amplification, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with iScript, SuperScript III (SSIII), and uMRT. (B) uMRT performs rolling circle reverse transcription at lower temperatures. 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with uMRT at room temperature (25 °C), 30 °C, and 42 °C. (C) Sanger sequencing results of concatemeric RT-PCR products by SuperScript III and uMRT. The circRNA sequence (top, green annotation) shows the circRNA junction (annotated with the orange triangle).

Article Snippet: In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT ® (uMRT), a highly processive reverse transcriptase.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) SuperScript III and uMRT were compared in their ability to reverse-transcribe larger circRNAs. The following circRNAs were used as input for the reverse transcription: lncATV (420 nt), circ-Znf609 (935 nt), and CVB3-EGFP (1596 nt). (B) Nanopore sequencing results of RT-PCR amplicons. Sequencing results across the junctions are shown (yellow highlighted region). Note that circ-lncATV and circ-Znf609 have identical splicing junctions due to the synthesis method. The whole sequence alignment is shown by the grey bar.

Article Snippet: In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT ® (uMRT), a highly processive reverse transcriptase.

Techniques: Reverse Transcription, Nanopore Sequencing, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) RT-PCR of circHIPK3 using uMRT, SuperScript III (SSIII), and SuperScript IV (SSIV). The expected RT-PCR product for circHIPK3 is 1,099 nt. (B) Method overview for detecting natural circRNAs. RNA extraction from HEK293T cells is treated with RNase R to remove linear RNA. (C) Nanopore sequencing results of the uMRT RT-PCR amplicon. Sequencing results across the junction are shown (yellow highlighted region). The whole sequence alignment is shown by the grey bars.

Article Snippet: In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT ® (uMRT), a highly processive reverse transcriptase.

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Nanopore Sequencing, Amplification, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with iScript, SuperScript III (SSIII), and uMRT. (B) uMRT performs rolling circle reverse transcription at lower temperatures. 3xFLAG (114 nt) circular RNA was used as a template for RT-PCR with uMRT at room temperature (25 °C), 30 °C, and 42 °C. (C) Sanger sequencing results of concatemeric RT-PCR products by SuperScript III and uMRT. The circRNA sequence (top, green annotation) shows the circRNA junction (annotated with the orange triangle).

Article Snippet: The method described here enables the straightforward validation of individual circRNA sequences, without the need for enrichment strategies or cloning, by using the highly processive UltraMarathonRT (uMRT) [17,18].

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) SuperScript III and uMRT were compared in their ability to reverse-transcribe larger circRNAs. The following circRNAs were used as input for the reverse transcription: lncATV (420 nt), circ-Znf609 (935 nt), and CVB3-EGFP (1596 nt). (B) Nanopore sequencing results of RT-PCR amplicons. Sequencing results across the junctions are shown (yellow highlighted region). Note that circ-lncATV and circ-Znf609 have identical splicing junctions due to the synthesis method. The whole sequence alignment is shown by the grey bar.

Article Snippet: The method described here enables the straightforward validation of individual circRNA sequences, without the need for enrichment strategies or cloning, by using the highly processive UltraMarathonRT (uMRT) [17,18].

Techniques: Reverse Transcription, Nanopore Sequencing, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Bio-protocol

Article Title: Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

doi: 10.21769/BioProtoc.5480

Figure Lengend Snippet: (A) RT-PCR of circHIPK3 using uMRT, SuperScript III (SSIII), and SuperScript IV (SSIV). The expected RT-PCR product for circHIPK3 is 1,099 nt. (B) Method overview for detecting natural circRNAs. RNA extraction from HEK293T cells is treated with RNase R to remove linear RNA. (C) Nanopore sequencing results of the uMRT RT-PCR amplicon. Sequencing results across the junction are shown (yellow highlighted region). The whole sequence alignment is shown by the grey bars.

Article Snippet: The method described here enables the straightforward validation of individual circRNA sequences, without the need for enrichment strategies or cloning, by using the highly processive UltraMarathonRT (uMRT) [17,18].

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Nanopore Sequencing, Amplification, Sequencing