NadPrep Total RNA-To-DNA Module

Catalog Number:

#1002401#1002402

NadPrep Total RNA-To-DNA Module is a reverse transcription module developed for NGS platform. This module offers an efficient solution for the generation of dsDNA fragments from 10-200 ng of Human total RNA from cells, tissues and FFPE samples.The dsDNA output can couple with the NadPrep DNA Library Preparation kit.

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Features

Versatile: Compatible with multiple RNA samples, including cells, fresh tissues and FFPE tissues.
High Performance: Ensure dsDNA library output with high efficiency and stability.
Friendly Workflow: The dsDNA output can directly couple with the NadPrep DNA Library Preparation kit without fragmentation steps.

Applications

Application	Sample type	Input recommended
RNA-Cap Sequencing	Cell line	10-200 ng
	Tissue	10-200 ng
	FFPE tissue	20-200 ng

Workflow

Performance

dsDNA yield from different amount of RNA input

Figure 1. dsDNA yield from different input amount of cell RNA (10 ng, 50 ng, 100 ng and 200 ng) using NadPrep Total RNA-To-DNA Module.

High Efficiency of Target Enrichment

Figure 2. Performance of Total RNA-Seq，rRNA-depleted RNA-Seq and RNAcap-Seq. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI). NanOnco Plus Research Panel v2.0 was used for RNAcap-Seq. A. RNAcap-Seq showed the lowest rRNA proportion in both K562 cell lines and FFPE tissues. B. RNAcap-Seq showed the highest enrichment efficiency for exon regions. C. RPKM values (in Reads per Kilobase of Exon per Million Reads) differ RNA-seq methods for libraries prepared from FFPE tissues. RNAcap-Seq can significantly enrich the targeted area for 20-50 times.

High Performance of Single Capture

Figure 3. Comparison of single capture and double capture with different panels on various NGS platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by different panels. A-B. Sequencing results on MGI platform (MGISEQ-2000, PE100); C-D. Sequencing results on Illumina® platform (Novaseq 6000, PE150). The results indicate that single capture can achieve an efficient enrichment similar to double capture.

Compatible with Multiple Sample Types

Figure 4. The effect of FFPE sample quality on RNA-Cap. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and were captured by NanOnco Plus Research Panel v2.0. A. The correlation between rRNA proportion and DV200. B. The correlation between on-target rate and DV200. For high quality FFPE samples (DV 200 > 60%), the rRNA ratio <10% and on-target rate > 80%; For highly degraded FFPE samples (DV 200 <30%), the rRNA ratio significantly increased with the capture performance declined. The DV 200 reveals the proportion of RNA fragments over 200 nucleotides.

Assessment of gDNA Contamination

Figure 5. Assessment of gDNA contamination in RNA samples. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and were captured by NanOnco Plus Research Panel v2.0. The specific targeted regions (known as the non-coding regions) are recommended to evaluate the DNA contamination. The threshold of contamination for RNA-Cap is required (10 read counts/Gb data, for instance).

Gene Fusion Detection

Figure 6. Fusion detection of FFPE RNA standard. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and captured by NanOnco Plus Research Panel v2.0. The test samples are gradient-diluted Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001), and the ordinate is the normalized Spanning Reads.

Compatible with MGI Platform and Illumina® Platform

Figure 7. Comparison of quality control parameters of RNA-Cap sequencing on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Research Panel v2.0 combined with Ext-RNA Control Panel. The quality control parameters of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from K562 cell lines and Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) reveal no significant difference, including A. on-target ratio, B. rRNA ratio, C. ERCC ratio and D. ERCC count.

Figure 8. Transcript coverage of RNA-Cap sequencing on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Research Panel v2.0 combined with Ext-RNA Control Panel. The transcript coverage of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from A. K562 cell lines and B. Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) reveal uniformity.

Figure 9. Comparison of RPKM on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Research Panel v2.0 combined with Ext-RNA Control Panel. The RPKM of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from A. K562 cell lines and B. Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) was compared.

For research use only, not for use in diagnostic procedures.

Package	Color of Tube Cap	Component	Volume 1002401 (24 rxn)	Volume 1002402 (96 rxn)
Box1		1st Strand Synth. Buffer	120 µL	460 µL
		Random Primer	30 µL	115 µL
		1st Strand Synth. Enzyme	60 µL	230 µL
		2nd Strand Synth. Buffer	810 µL	2×1550 µL
		2nd Strand Synth. Enzyme	90 µL	345 µL
	/	Nuclease Free Water	2.5 mL	8 mL
Box2	/	NadPrep SP Beads	2.5 mL	10 mL

Is this module suitable for the total RNA sequencing?

The library for RNA-Seq can be prepared with this module coupled with the rRNA removal module. The NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB, Cat # E7400L) is recommended.

Why DV 200 is used to classify the RNA from FFPE tissue?

The RIN of RNA extracted form FFPE tissue is generally small, of which the value is mostly around 2.0. While the DV 200 differs greatly among the RNA samples, which indicates the actual degradation degree of RNA extracted from FFPE tissue. Therefore, we highly recommend DV 200 as the QC reference of RNA from FFPE tissue.

Is this module suitable for the highly degraded RNA from FFPE tissues?

This module is suitable for most of RNA samples from cells and tissues. As for the highly degraded RNA from FFPE tissues (DV 200 < 30%), the efficiency of dsDNA targeting declines greatly. When using RNA from FFPE tissues for targeted sequencing, it is recommended to augment the RNA input to improve the capture efficiency.

How to improve the dsDNA output from total RNA?

Make sure to proceed the RNA-related steps using RNase-free consumables in the area without RNase contamination. Furthermore, additional purification step by using NadPrep SP Beads is necessary for wiping off the residual impurities, which may inhibit the subsequent enzymatic reaction, including chelating agent, guanidine salt, phenol, ethanol, etc.