HiPure Total RNA Kit

The HiPure Total RNA Kit provides a single-column silica membrane workflow for purification of large RNA molecules (>200 nt) from cultured cells and soft tissues. The method relies on chaotropic lysis followed by selective RNA adsorption on a silica membrane and sequential washing steps to remove proteins, salts and other contaminants.

The streamlined workflow is widely used for routine RNA preparation in molecular biology laboratories and supports downstream applications such as RT-PCR, qPCR and gene expression analysis. The protocol is suitable for standard laboratory workflows requiring consistent RNA purification from cultured cells or soft animal tissues.

Within the Magen RNA extraction systems, laboratories requiring genomic DNA removal during RNA purification may refer to the HiPure Total RNA Plus Kit (R4111), while automated RNA purification workflows can be implemented using the MagPure Universal RNA Kit (IVD3020).

Workflow

Workflow Overview

The HiPure Total RNA Kit uses a single-column silica membrane workflow for routine purification of total RNA from cultured cells, soft tissues, plant material, yeast and bacterial samples. Following sample disruption and lysis in RTL Lysis Buffer, RNA binding conditions are established by adding RNA Binding Buffer. The prepared lysate is then loaded directly onto the RNA column, where RNA is captured on the silica membrane and recovered through washing, drying and elution steps. For downstream applications that are highly sensitive to residual DNA, optional on-column DNase treatment can be incorporated into the workflow.

Sample Handling Logic

This workflow is designed as a direct RNA purification route, with the main sample-dependent variation concentrated in the disruption and lysis stage. Soft tissue and cultured cell samples can usually enter the workflow after homogenization and clarification, while plant material, yeast and bacterial samples may require stronger mechanical disruption before column loading. Once lysis and binding preparation are complete, the downstream single-column purification steps remain consistent, providing a straightforward format for routine total RNA recovery.

Time and Workflow Characteristics

Under typical manual operation, the overall workflow is usually completed within about 25–45 minutes, depending mainly on sample type, disruption method and lysate viscosity. The workflow is shorter and operationally simpler than dual-column or phase-separation routes because RNA binding, washing, drying and elution are performed on a single RNA column. For detailed step-by-step conditions, workflow guidance and estimated processing times, please refer to the Workflow Note in the Download section.

Specifications

Principle

HiPure RNA technology simplifies total RNA isolation. Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the HiPure silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment. Pure, concentrated RNA is eluted in water.

Technical Validation

HiPure Total RNA Kit was evaluated using animal and plant tissue samples to assess RNA yield, purity and integrity in a silica membrane spin-column workflow. The validation included manual extraction from 20 mg animal tissues and 100 mg plant tissues, with purified RNA analyzed by Nanodrop and agarose gel electrophoresis.

In animal tissue testing, RNA was extracted from kidney, spleen and liver samples and compared with a reference column-based RNA extraction workflow. From 20 mg tissue input, RNA yields ranged from 56–60 µg for kidney, 50–52 µg for spleen, and 81–105 µg for liver samples. The measured A260/280 values were 2.06–2.11, and A260/230 values were generally within 2.06–2.23, supporting recovery of RNA with suitable purity for routine downstream analysis.

Plant tissue extraction was further evaluated using pepper leaves, pumpkin leaves and Pachira macrocarpa leaves. From 100 mg plant input, the kit produced RNA yields of 55.47–70.31 µg from pepper leaves, 76.26–77.20 µg from pumpkin leaves and 46.13–50.31 µg from Pachira macrocarpa leaves. A260/280 values were 1.97–1.99, with most A260/230 values above 2.2 in the tested R4011 samples.

Electrophoresis analysis showed intact RNA band patterns from both animal and plant samples, supporting RNA integrity after extraction. The kit format also supports optional on-column DNase treatment for applications sensitive to residual genomic DNA, and the purified RNA is suitable for downstream workflows such as RT-PCR, Northern blotting, poly(A)⁺ RNA purification, nuclease protection and in vitro translation.

Kit Contents

Storage and Stability

The Kit can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions.

For a broader view of Magen tissue and cell RNA extraction routes, the following resources may help place this product within the complete workflow system.