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Title: Is the NadPrep EZ RNA Library Preparation Kit suitable for severely degraded FFPE samples?

Yes. It is. NadPrep EZ RNA Library Preparation Kit is suitable to most RNA samples from different grades and sources (cells, fresh tissues etc.). For FFPE-derived RNA samples with severe fragmentation (DV200 < 20), reverse transcription efficiency may be reduced, and it is recommended to increase the no. of PCR cycles as appropriate. When performed targeted capture on FFPE-derived RNA samples, consider increasing the RNA input to improve capture efficiency.

Title: Which sample types are the NadPrep EZ RNA Library Preparation Kit applicable for?

NadPrep EZ RNA Library Preparation Kit is widely suitable for the libraries prepared from total RNA with different grades, poly(A)-enriched mRNA, and rRNA-depleted RNA. It should be noted that the mRNA content in total RNA from different sources varies greatly. If the initial input amount of total RNA is too low, sufficient mRNA may not be obtained to prepare a high-quality library.

Title: What’s the difference between non-stranded and stranded libraries from NadPrep EZ RNA Library Preparation Kit?

RNA-Seq library preparation generally falls into two types: non-stranded (general) and stranded (strand-specific). The core difference is whether the library retains transcript directional information. Among them, stranded libraries retain the transcript directional information during the preparing process, and sequencing and downstream analysis can determine whether the transcripts originate from the sense or antisense strands of DNA. However, non-stranded libraries lose directional information during cDNA synthesis, and sequencing data cannot directly indicate transcript directions. Compared with general RNA-Seq, strand-specific RNA-Seq provides more accurate transcript quantification, clearer gene structures, identification of antisense transcripts, and discovery of novel transcripts. Therefore, stranded RNA-Seq is crucial for studies of gene structure and expression regulation. To obtain transcript direction information, stranded libraries should be preferred.

Title: Can RNA samples be directly used for library preparation with this kit?

No. This kit is compatible only with gDNA or cDNA as initial samples. For RNA samples, reverse transcription to generate cDNA is required prior to library preparation.

Title: What is the recommended input range? How to handle inputs exceeding this range?

This kit supports 50-2,000 ng of gDNA or cDNA. For input amount exceeds 2,000 ng, split the sample into multiple reactions to maintain amplification efficiency.

Title: What is the insert size of this kit? How to select sequencing read length?

The main peak of PCR products by using this kit is ~270 bp. PE150 sequencing is recommended for high-quality coverage.

Title: What is the recommended sequencing data volume for IGTR immune repertoire analysis?

A minimum of 0.3 Gb is recommended to detect clones at 0.01% abundance with an input of 200 ng. Increase data volume to enhance sensitivity for low-frequency clones.

Title: Is this kit suitable for minimal residual disease (MRD) monitoring?

Yes. This kit includes IG Primer Mix and TR Primer Mix with gene-specific primers provided in separate tubes, allowing flexible combination in a single amplification reaction. With its high sensitivity, the kit meets the requirements for MRD monitoring technology development and clinical applications, making it ideal for low-frequency variant detection scenarios.

Title: Is there any difference in the capture performance of libraries prepared from RNA samples using the NadPrep RNA & DNA Library Co-Preparation Module and NadPrep Total RNA-To-DNA Module conjunction NadPrep DNA Library Preparation Module?

FAQ-Q7


Figure 2. Correlation of ERCC expression profiles in captured libraries from RNA samples processed through different library preparation protocols. A. NadPrep RNA & DNA Library Co-Preparation Module; B. NadPrep Total RNA-To-DNA Module conjunction NadPrep DNA Library Preparation Module.

Note: The samples originated from K562 cell line RNA with an initial input of 50 ng; the pre-library input amount was 200 ng.


Title: Is there a difference in performance between the NadPrep RNA & DNA Library Co-Preparation Module and the NadPrep EZ DNA Library Preparation Module v2 when preparing libraries from DNA samples?

FAQ-Q6


Figure 1. Library yield and capture performance of DNA samples using different library preparation protocols. A. Library Yield; B. Mappability.

Note: The sample originated from 50 ng of Human Genomic DNA Standard (Promega, G1521). Sequencing mode was Illumina NovaSeq 6000 platform, PE150.