Single Cell Gene Expression: Direct Lysate Analysis
Direct cDNA Cell Lysis Buffer, RT-PCR & Real-Time PCR Assays
Introduction
Gene expression analysis often requires RNA preparation followed by cDNA synthesis and PCR or real-time PCR analysis. However, when working with very small sample amounts, such as single cells, FACS-sorted cells, laser capture microdissection samples, cultured cells in 96-well plates, stem cells, neurons, or rare clinical samples, traditional RNA extraction can lead to significant sample loss.
Signosis provides single-cell gene expression tools designed to simplify low-input sample analysis. Our Direct Cell Lysis Buffer allows researchers to prepare cell lysates directly from small numbers of cells without RNA purification. The resulting lysate can be used for downstream reverse transcription, PCR, real-time PCR, or cDNA plate array analysis.
Principle
Signosis Direct Cell Lysis Buffer lyses small numbers of cells under conditions compatible with downstream reverse transcription. Instead of isolating RNA, the prepared cell lysate is added directly to the reverse transcription reaction to generate cDNA. The resulting cDNA can then be analyzed by endpoint PCR, real-time PCR, or cDNA plate array assays to measure gene expression from very limited starting material.
Figure 1: Sensitive RT-PCR from Direct Cell Lysates
Caption for Figure 1
A: Cells were lysed using Signosis Direct cDNA Cell Lysis Buffer or a competitor buffer, then analyzed by RT-PCR for β-actin after 30 cycles.
B: Genomic DNA contamination was evaluated by amplifying β-actin directly from cell lysate without reverse transcription (Lane 1) and from cDNA generated from the same lysate (Lane 2).
Figure 2: Reduced Genomic DNA Contamination
Caption for Figure 2
PCR products from lysates prepared with Signosis Direct cDNA Cell Lysis Buffer were compared with RNA prepared using TRIzol without DNase treatment. Using intron-spanning primers, genomic DNA and reverse-transcribed mRNA products could be distinguished. TRIzol-prepared samples showed both genomic DNA and mRNA bands, while Signosis lysates showed only the expected mRNA-derived band, indicating reduced genomic DNA contamination.
Product Options
Direct Cell Lysis Buffer
Signosis Direct Cell Lysis Buffer is designed to lyse cells quickly and gently while maintaining RNA compatibility for downstream cDNA synthesis. It eliminates the need for traditional RNA extraction and allows researchers to proceed directly from cell lysate to reverse transcription.
Single Cell RT-PCR Assay Kit
The Single Cell RT-PCR Assay Kit provides a complete endpoint PCR workflow for detecting target gene expression from single-cell or low-cell-number lysates. It is ideal for researchers who need sensitive transcript detection without RNA isolation.
Single Cell Real-Time RT-PCR Assay Kit
The Single Cell Real-Time RT-PCR Assay Kit is designed for quantitative gene expression analysis from direct cell lysates. It is compatible with standard real-time PCR instruments and allows researchers to monitor gene expression levels using Ct-based quantification.
Benefits
No RNA Purification Required
Cell lysates can be used directly for cDNA synthesis, helping avoid sample loss from column-based or precipitation-based RNA purification.
Reduced Sample Loss
By eliminating RNA extraction, the workflow helps preserve rare or low-abundance transcripts that may otherwise be lost during purification.
Designed for Low-Input Samples
The system is suitable for single cells, small cell populations, sorted cells, microdissected tissue samples, and other scarce biological materials.
Simple and Efficient Workflow
The direct lysis approach reduces hands-on time, minimizes pipetting steps, and supports more consistent results when working with small sample volumes.
Compatible with Multiple Downstream Applications
Prepared lysates can be used for reverse transcription followed by PCR, real-time PCR, or cDNA plate array analysis.
