LeXPrep EZ RNA Library Preparation Module

Low library yields are typically caused by three primary factors:

• RNase Contamination: RNA is highly susceptible to degradation. Solution: Maintain a strictly nuclease-free environment. Use RNase-free certified consumables and perform all steps in a dedicated clean space to prevent degradation that inhibits reverse transcription.
• Sample Impurity: Residual contaminants (e.g., phenol, ethanol, guanidine salts, or chelators) can inhibit enzymatic activity. Solution: We recommend an additional RNA purification step using LeXPrep SP Beads (2X ratio) and eluting in Nuclease-Free Water to ensure optimal enzyme performance.
• Low Initial Quality: Severely degraded samples naturally produce lower yields. Solution: For low-quality or highly fragmented samples, increase the number of PCR enrichment cycles as recommended in the technical manual.

Yes. The LeXPrep EZ RNA Library Preparation Kit is engineered for high versatility and is compatible with various sample types, including cells, fresh tissues, and FFPE-derived RNA.

For highly fragmented FFPE samples (e.g., DV200 < 20), reverse transcription efficiency may be reduced. In these cases, we recommend:

• Increasing the number of PCR cycles.
• Increasing the initial RNA input amount—especially for targeted capture applications—to maximize complexity and capture efficiency.

The LeXPrep EZ RNA Library Preparation Kit is widely applicable to various starting materials, including:

• Total RNA (from multiple species and grades)
• Poly(A)-enriched mRNA
• rRNA-depleted RNA

Note on Input: Because mRNA content varies significantly between different tissue types and sources, ensure that your total RNA input is sufficient to yield the required amount of mRNA for high-complexity library construction.

The primary difference lies in the preservation of the transcriptional directional information:

• Stranded (Strand-Specific) Libraries: These retain the original orientation of the transcript. This allows researchers to determine whether a read originated from the sense or antisense strand. Stranded RNA-Seq is essential for accurate gene quantification, identifying antisense transcripts, and resolving overlapping gene structures.
• Non-Stranded (General) Libraries: Directional information is lost during second-strand cDNA synthesis. While suitable for basic expression profiling, non-stranded libraries cannot distinguish between sense and antisense transcription.

Recommendation: For comprehensive studies of gene regulation and structure, stranded library preparation is the preferred gold standard for higher-resolution data.