All insights start with the sample

Explore the RNA universe!

RNAs are crucial molecules with diverse functions in the cell. Messenger RNAs (mRNA) serve as templates for protein synthesis, while ribosomal RNAs (rRNA) are components of ribosomes. Transfer RNAs (tRNA) deliver amino acids for protein synthesis. Non-coding RNAs, including long noncoding RNA (lncRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), short interfering RNA (siRNA), and piwi-interacting RNA (piRNA), play roles in gene expression regulation. RNAs are also involved in riboprotein complexes for RNA processing.

A mammalian cell typically contains 10-30 pg of total RNA, with differentiated cells having less. tRNAs and rRNAs constitute the majority of cellular RNA, while mRNA accounts for 3-7%, varying by cell type and physiological state. RNA analysis provides insights into an organism's gene expression profile. Reliable results in RNA profiling, such as real-time RT-PCR or transcriptome sequencing, depend on the quality of the RNA sample and its processing. QIAGEN offers technologies for stabilization and disruption to ensure success in RNA experiments.

Key RNA types include:

• Small (short) interfering RNA (siRNA)
• Ribonuclease P (RNase P)
• Small nucleolar RNA (snoRNA)
• Y RNA
• Small nuclear RNA (snRNA)
• Ribonuclease MRP (RNase MRP)
• MicroRNA (miRNA)
• Ribosomal RNA (rRNA)
• Long non-coding RNA (lncRNA)
• Messenger RNA (mRNA)
• Transfer RNA (tRNA)
• Signal recognition particle RNA (SRP RNA)
• Piwi-interacting RNA (piRNA)
• Circulating cell-free RNA (ccfRNA)
• Exosomal RNA (exRNA)

For a detailed poster, visit www.qiagen.com/RNA-Poster.

RNA Distribution and Achieving Reproducible Results

RNA Distribution:

• By Mass: rRNA constitutes 80-90%, tRNA 10-15%, mRNA 3-7%, and other ncRNAs 0.03-1%.
• By Number of Molecules: tRNA is the most abundant, followed by rRNA, with mRNA and other ncRNAs present in smaller quantities.

The scientific community faces a growing concern regarding research reproducibility. High-quality, reproducible studies are foundational for scientific progress. However, many scientists struggle to reproduce results, leading to wasted resources and damaged credibility. QIAGEN emphasizes standardizing sample purifications and implementing quality control (QC) checks to enhance RNA experiment reliability. High-quality sample stabilization and purification chemistries protect RNA from degradation and remove interfering contaminants, ensuring accurate downstream assay results. QC measures can identify sample quality variations, allowing for timely corrective actions and processing of only the highest quality samples.

Sample Collection and Stabilization

Harvesting biological samples can alter RNA expression profiles due to ex vivo gene induction or downregulation and RNA degradation by RNases. Immediate stabilization of cellular RNA is critical for accurate gene expression analysis, capturing a snapshot of the true profile at the time of collection.

Traditionally, samples were frozen in liquid nitrogen and stored at -80°C. However, liquid nitrogen is hazardous and not always accessible. QIAGEN offers convenient, easy, and safe sample handling and preservation solutions at room temperature, detailed in product selection guides. For integrated sample collection, stabilization, and purification, QIAGEN's PAXgene® Systems are available. Learn more at www.PreAnalytiX.com.

QIAGEN RNA stabilization technologies enable:

• Immediate RNA stabilization, preserving the RNA profile and depleting contaminants.
• Safe sample handling and preparation at room temperature, eliminating the need for liquid nitrogen or dry ice.
• Convenient sample shipping without temperature fluctuation impact on RNA integrity.
• Long-term sample archiving without RNA degradation.

Illustration of mRNA changes: After sample harvest, mRNA levels can be affected by induction or enzymatic degradation/down-regulation.

Gel Electrophoresis Example: Figure 3 shows purified RNA from stabilized rat kidney samples, demonstrating intact RNA bands, compared to unstabilized samples which show degraded RNA.

Sample Disruption and Homogenization

Effective sample disruption and homogenization are essential for RNA purification. Incomplete processes reduce RNA yields and can bias profiling. Disruption makes RNA accessible to RNases, so it must be performed under conditions that denature RNases.

QIAGEN offers various disruption and homogenization technologies, including lysis buffers, QIAshredder spin columns, TissueRuptor® II, and Tissuelyser II and LT systems for mechanical disruption of tough samples.

• Disruption breaks down tissue structures, cell walls, and membranes to release RNA.
• Homogenization reduces sample viscosity for easier purification.

Disruption and Homogenization Methods (Table 1)

QIAGEN Disruption Systems (Table 2)

Disruption and homogenization using bead mills

Bead mills agitate samples at high speed with beads, causing hydrodynamic shearing and crushing for disruption and homogenization. Efficiency depends on bead size/composition, buffer-to-bead ratio, starting material amount, agitator speed, and disintegration time.

Recommended beads:

• Bacteria: 0.1 mm glass beads
• Yeast and unicellular animal cells: 0.5 mm glass beads
• Animal and plant tissues: 3-7 mm stainless steel beads

Glass beads are recommended for bacteria, while stainless steel beads are effective for larger tissues. Tungsten carbide beads can be used for plant samples.

Disruption and homogenization using rotor-stator adapters

Rotor-stator homogenizers disrupt and homogenize tissues and cell lysates quickly (5-90 seconds) using high-speed rotation and mechanical shearing. To avoid foaming:

• Use a properly sized vessel.
• Keep the homogenizer tip submerged.
• Hold the immersed tip to one side of the tube.

RNA Isolation

Selecting an appropriate RNA isolation method is crucial for maintaining RNA integrity and removing contaminants that can inhibit downstream applications like RT-PCR and NGS. QIAGEN's portfolio of RNA purification kits addresses various sample types and challenges.

Spin-column technology

QIAGEN spin-column protocols involve four steps:

• Lyse: Efficient lysis releases RNA and inactivates RNases.
• Bind: Optimal conditions bind RNA to a silica membrane.
• Wash: The membrane is washed to remove contaminants.
• Elute: RNA is released from the membrane for downstream assays.

Benefits of QIAGEN RNA isolation kits:

• High efficiency in contaminant removal, yielding high-purity RNA.
• Dedicated products optimized for diverse sample types.
• Maximal yield, providing an unbiased snapshot of RNA composition.

Typical RNeasy Procedure: The process involves sample lysis, homogenization, ethanol addition, binding RNA to the RNeasy membrane, washing, and eluting the purified RNA.

RNA Sample and Binding Capacities

QIAGEN offers various spin column sizes for RNA isolation, each with different sample and binding capacities (Table 3). RNeasy Kits are designed for RNA larger than 200 nucleotides, while miRNeasy Kits isolate total RNA, including small RNAs from approximately 18 nucleotides upwards. miRNeasy kits can also separate miRNA and mRNA fractions.

Table 3: Sample and binding capacities of different spin column sizes

Figure 5: Efficient copurification of miRNA and RNA from a wide range of tissues. This bar chart illustrates the efficient copurification of total RNA, including miRNA, from rat tissues using the miRNeasy 96 Kit, with quantification of miR-16 and PGK1 via qRT-PCR.

Did you know? QIAGEN invented spin-column technology over 30 years ago, revolutionizing nucleic acid purification.

High-purity RNA from FFPE tissue sections

Purifying nucleic acids from FFPE tissue sections presents challenges due to crosslinking and fragmentation. QIAGEN's RNeasy and miRNeasy FFPE Kits utilize special lysis and incubation conditions, including Proteinase K, to efficiently release RNA and reverse crosslinking. RNA is treated with DNase to remove DNA fragments, ensuring high-quality total RNA for downstream applications.

The PAXgene Tissue System offers simultaneous, crosslinking-free stabilization of molecular content and preservation of histomorphology, allowing samples to be embedded in paraffin for histological studies and subsequent RNA/miRNA isolation.

Figure 6: Recovery of all usable RNA. This electropherogram compares RNA recovery from FFPE rat liver samples using the RNeasy FFPE Kit versus kits from other suppliers, showing QIAGEN's superior performance.

Cell-free RNA purification from serum or plasma samples

RNA released by apoptotic or necrotic cells is often degraded by RNases in plasma and serum. However, cell-free miRNA bound to proteins like Ago2 and RNA within exosomes and extracellular vesicles (EVs) are protected and relatively stable. These circulating RNAs can serve as biomarkers but their purification is challenging due to low abundance and high RNase levels.

Figure 7: Illustration of circulating cell-free RNA in serum or plasma. This diagram shows cell-free miRNA bound to Ago2 protein and extracellular vesicles containing cell-free miRNA and mRNA.

The miRNeasy Serum/Plasma Advanced Kit efficiently purifies total cell-free RNA from serum and plasma. The exoRNeasy Serum/Plasma Kits specifically isolate vesicular RNA.

Removal of genomic DNA contamination

While RNeasy and miRNeasy kits inherently remove a significant portion of genomic DNA (gDNA), trace amounts can affect sensitive applications like real-time RT-PCR. QIAGEN offers various technologies to remove residual gDNA.

Figure 8. Overview of QIAGEN gDNA removal technologies. This diagram illustrates the workflow for RNA purification, cDNA synthesis, and the role of gDNA removal steps to yield gDNA-free RNA for downstream applications.

Co-purification of RNA and other analytes

For systems biology research requiring parallel analysis of multiple analytes from the same sample, QIAGEN's AllPrep® Kits provide a streamlined protocol to purify RNA, DNA, or proteins simultaneously, eluting each biomolecule in a separate fraction. This maximizes yields and ensures reliable genomic, transcriptomic, and proteomic analyses. AllPrep kits can be combined with Allprotect® Tissue Reagent for enhanced stabilization.

The QIAamp® ccfDNA/RNA Kit enables co-purification of cell-free DNA and RNA from serum or plasma, facilitating analysis of mutations and improving detection sensitivity for low-frequency tumor mutations.

Figure 9. Example of the AllPrep procedure. This diagram shows the workflow for isolating different analytes from a sample, with separate eluates for RNA and DNA.

Purification of viral nucleic acids

Concentrating dilute viral RNA and DNA from plasma and serum is a key challenge. QIAamp virus kits are designed for viral nucleic acid purification from volumes ranging from 140 µl to 10 ml, utilizing silica-membrane spin columns for efficient isolation. These kits offer increased sensitivity for viral-load monitoring.

Table 4: Example of viral RNA recovery

Automate your RNA isolation

Routine RNA purification can be tedious and prone to human error. QIAGEN's automated RNA purification systems support low to high throughputs, from single samples to 96-well formats. These systems, combined with QIAGEN chemistries, ensure reproducible purification of high-quality RNA. Dedicated kits are available for automated platforms.

QIAGEN Instrument Services offers benefits such as flexible service support, dedicated application support, and tailor-made training programs.

Manual and Automated RNA Purification Kits

Table 8: Kits for manual, high-throughput RNA purification

Table 9: Automated RNA purification kits

For more information on QIAGEN automation solutions, contact your QIAGEN sales person or visit www.qiagen.com/automation.

RNA quality control

Reliable results in downstream assays depend on RNA quality, assessed by Quantity, Purity, and Integrity. QIAGEN's QIAxpert® and QIAxcel® Advanced Systems provide simple steps for rigorous RNA quality control.

Measurement of RNA quantity and purity

RNA concentration is measured via absorbance at 260 nm (A260). Purity is estimated using the A260/A280 ratio, with 1.8–2.1 at pH 7.5 indicating high purity. The A260/A230 ratio, ideally around 2, can indicate contamination by salts like guanidine thiocyanate. The QIAxpert spectrophotometer uses Spectral Content Profiling (SCP) to differentiate DNA, RNA, and other contaminants.

Figure 11. Spectral content profiling discriminates components of complex samples. This shows spectral plots for a pure RNA sample and a sample spiked with gDNA, illustrating the ability to resolve contributing absorbance lines.

Measuring RNA integrity

RNA integrity is assessed by electrophoretic signatures, which reflect the size distribution of RNA subpopulations, including ribosomal RNAs (5S, 18S, 28S). Denaturing agarose gel electrophoresis or capillary electrophoresis systems like QIAxcel Advanced can be used. RNA Integrity Score (RIS) or RNA Integrity Number (RIN) values above 8 indicate high-quality RNA. Sample quality is often influenced by sample preservation.

Table 5: rRNA sizes from various sources

Figure 12. Analysis of total RNA purified from S. pombe. This presents gel image and electropherogram results for RNA analysis.

Product selection guide

Table 6. Products for sample collection and stabilization

Table 7. RNA isolation selection guide

Table 8. Kits for manual, high-throughput RNA purification

Table 9. Automated RNA purification kits

Table 10. Kits for co-purification of RNA and other analytes

Table 11. QIAamp solutions for isolation of viral nucleic acids

Table 12. Solutions for in vitro diagnostic applications