Qiagen rna extraction kit manual

However yield is highly donor-dependent, and in some cases higher or lower yields may be achieved. To prevent low yields due to underfilling of the tube, see the phlebotomy FAQ and the blood collection demonstration video. A blood collection set is required to be used with the PAXgene Blood RNA Tube to eliminate the possibility of patient contact with the reagent in the tube, due to backflow of blood, when used in accordance with the instructions for use. Long term archiving studies are ongoing.

Waste from the sample preparation, such as supernatants from centrifugation steps in the RNA purification process, is to be considered potentially infectious. Before disposal, the waste must be autoclaved or incinerated to destroy any infectious material. Disposal must follow official regulations. Possible reasons for blood draws with lower than expected blood volume include: 1 The phlebotomist has not waited enough time for the blood to stop flowing into the tube. Reorder now! Reorder from your past orders in just one click.

Order by Quote. Quote Number. Add quote number from your quote document. Customer Number. Add customer number from your quote document. To remove a quote go to the Cart. View Quote Example. Catalog Number. Looking for a quick way to design experiments? Try the Workflow Configurator. A convenient tool to build experimental workflows and find products to match your needs.

Log Out. Show More. Log in to see your account pricing. Reproducible and repeatable RNA purification. Repeatability and reproducibility of RNA yield. Reproducibility between users. Reproducibility between kit lots. RNA yield and purity — automated processing. Reproducibility between automated and manual protocols. Supporting data and figures Reproducible and repeatable RNA purification. In vitro diagnostic medical device. PDF 1MB. For collection, transport, and storage of whole blood and stabilization and purification of intracellular RNA.

Scientific Posters 4. PDF KB. Guenther et al. PDF 7MB. PDF 81KB. Guenter et al. Safety Data Sheets 1. Safety Data Sheets EN.

Application Notes 2. It has successfully been tested for Reverse Transcription in bacteria as well. The RNeasy 96 Universal Tissue Kit enables high-throughput purification of RNA from any animal or human tissue sample, including difficult-to-lyse fibrous and fatty tissues.

Please refer to the Selection guide for RNA isolation for all sample types to find the optimal solution for your sample source.

The exact composition of Buffer RLT is confidential. This buffer is a proprietary component of RNeasy Kits. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane.

Buffer RLT can be purchased separately cat. RNA in tissues is not protected after harvesting until the sample is treated with RNAprotect Tissue Reagent , flash frozen, or disrupted and homogenized in the presence of RNase-inhibiting or denaturing reagents. Frozen tissue should not be allowed to thaw during handling or weighing, but cell pellets can partially thaw enough to allow them to be dislodged by flicking.

The relevant procedures should be carried out as quickly as possible. Frozen samples are stable for months. Please review the instructions in the relevant RNeasy Handbook carefully for best results.

The respective ribosomal species should appear as sharp bands on the stained gel. If the ribosomal bands are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA sample has suffered major degradation during preparation. It is not recommended for cultured cells because it may be difficult to pellet and remove them from the reagent prior to RNA isolation see FAQ This reagent is for immediate RNA stabilization in sorted or cultured cells with no need to remove medium.

Ribonucleases are the principal threat to any RNA isolation procedure. In addition, copurification of inhibitory contaminants is a major problem when isolating RNA from certain tissue sources.

To minimize the threat, gloves should be worn at all times, and special care must be taken to use RNase-free reagents and labware. It is very important to use a sufficient amount of lysis buffer during RNA isolation.

It is more challenging to isolate high-quality RNA from tissue samples than from cultured cells, especially those tissues containing high levels of RNase, or difficult-to-homogenize tissues.

Examples of such tissues include liver, heart, skin, and conjunctive tissues. Many tissue samples also contain difficult-to-remove contaminants such as polysaccharides, collagen, fats, lipids or fibrous components that may interfere with subsequent enzymatic reactions if not removed from the RNA preparation.

This ratio provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV range, such as protein. Be sure to calibrate the spectrophotometer with the same solution. However, values up to 2. For details on how the pH influences nucleic acid purity measurements, please review the reference ' Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity ', by Wilfinger WW, Mackey K, Chomczynski P, Biotechniques.

The QIAshredder is a unique biopolymer shredding system in a microcentrifuge spin-column format. It homogenizes cell or tissue lysates to reduce viscosity. Homogenization shears the high-molecular weight genomic DNA and other high-molecular-weight cellular components to create a homogenous lysate. The QIAshredder is chemically inert and will not bind nucleic acids.

It cannot replace tissue disruption or enzymatic cell wall lysis by mechanical and chemical methods, respectively. Both, efficient cell wall disruption and lysate homogenization are fundamental for successful RNA isolation from all types of samples.

Once complete disruption has been achieved, the QIAshredder Homogenizer can be used in place of needle and syringe, or rotor-stator homogenization. For further information regarding sample disruption and homogenization please refer to the 'Disruption and homogenization of starting materials' section in the RNeasy Mini Handbook , and see FAQ RNA has a high degree of secondary structure that needs to be resolved or denatured before running the sample out on a gel.

A formaldehyde gel needs to be used to disrupt the secondary structure and eliminate a ladder effect. Some banding pattern may remain due to the presence of mRNA transcripts of different lengths specific for the respective cell or tissue type. Complete disruption of cell walls and plasma membranes of cells and organelles is absolutely required to release all RNA contained in a sample. Different samples require different methods to achieve complete disruption.

Please refer to the section 'Disruption and homogenization of starting materials' in the RNeasy Mini Handbook. Incomplete disruption results in significantly reduced RNA yields. Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption. Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate.

Please note that the reaction must be cleaned up after the off-column DNase digest to remove the enzyme and buffer RDD, which will interfere with subsequent RT reactions.

To obtain a concentration of 1. Vortex briefly and keep on ice until use. In comparison to Buffer RLT of, e. These buffers contain guanidine thiocyanate, which can form highly reactive compounds when combined with acidic solutions. Acetone should be used instead to precipitate protein from RLT Plus lysates. In general, we always provide extra volume of buffers in our kits to account for pipetting errors and such. If you are left with extra buffers after using up all the columns in a kit, please refer to the Material Safety Data Sheet for respective kit to dispose off any unused buffers.

The filter can only tolerate low centrifugal forces not sufficient to shear genomic DNA. No, our standard RNeasy Mini Kit will not be discontinued. Pancreas is very high in RNases. Therefore, it is important to minimize the time between harvesting the tissue and snap freezing or stabilization in RNAprotect Tissue Reagent.

Tissue-Tek O. It mainly consists of glycols and synthetic resins. Using the O. Most cryosections are fixed using non-crosslinking agents. We strongly recommend removing as much of the embedding compound as possible prior to RNA extraction from the sections. In case crosslinking agents e. Special lysis and incubation conditions reverse formaldehyde modification of RNA for improved results in downstream application.

The crosslinking causes the RNA to break, resulting in overall smaller molecules, which look like a smear when analyzed on a formaldehyde gel. Our RNeasy buffers are subjected to stringent quality-control tests to ensure that they are indeed RNase-free. The efficiency of downstream applications depends strongly on the purity of the RNA sample used.

In our experience, the increased absorbance at nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.

In summary, we found that concentrations of guanidine thiocyanate of up to mM in an RNA sample do not compromise the reliability of downstream applications.

The RNA can be directly applied to the Agilent Bioanalyzer, since it is being denatured in the final protocol steps of these isolation procedures. Load the lysate onto the column in successive aliquots in step 5 of the protocol. Pellet cells by centrifugation. Caution: Cells might lyse. Note that the above steps are suggestions, rather than official protocol recommendations.

Please try a "pilot" run on a test sample first. In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e. Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase. Always be sure to calibrate the spectrophotometer with the same solution.

Please see the Appendix sections in the RNeasy handbooks for additional information. BioTechniques 22, Although the lysis and binding buffers in QIAamp, DNeasy, and RNeasy kits contain chaotropic agents that can inactivate some biohazardous material, local regulations dictate the proper way to dispose of biohazards.

DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Guanidine hydrochloride in the sample-preparation waste can form highly reactive compounds when combined with bleach. When working with RNA, care must be taken to avoid degradation by RNases, which are extremely stable and active. Intracellular RNases are released during the lysis step of the RNA isolation procedure and must be rapidly and thoroughly inactivated to obtain high-quality RNA.

In combination with the strong, but temporary denaturing effects of guanidinium isothiocyanate GITC contained in buffer RLT of the RNeasy Kits , any RNases present in the material to be extracted from will be completely inactivated. For more information on compatible kits and sample types, see our Selection Guide for RNA purification. The exact composition of Buffer RW1 is confidential. Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, fatty acids etc.

At the same time, RNA molecules larger than bases remain bound to the column. Buffer RWT should be used instead. The RNeasy Mini Kit Handbook contains a standard and an abbreviated protocol using enzymatic lysis, and one protocol using mechanical disruption. The enzymatic protocols employ zymolase and lyticase, and the mechanical disruption protocols employ glass beads and a beadmill for cell wall disruption. We recommend adding 20 ng of carrier RNA to the cell lysate before loading it onto the RNeasy membrane.

Reverse-transcription reactions typically contain a large excess of oligo-dT, and the small amounts of poly-A used as carrier RNA are insignificant in comparison. The RNeasy Micro procedure uses a novel technology to purify RNA from small amounts of tissues or cells as little as 1 cell.

Note, however, that carrier RNA has to be avoided in the RNA purification procedure as it may affect specific amplification of transcript sequences.

Degraded RNA cannot be used for amplification. The concentration of RNA isolated with RNeasy Kits can be determined by measuring the absorbance at nm A in a spectrophotometer. Absorbance readings should be greater than 0. This relationship is valid for measurements in water. Therefore, dilute RNA in water to quantify it spectrophotometrically. An example of the calculations involved in RNA quantification is shown below.

Use the buffer in which the RNA is diluted to zero the spectrophotometer:. Reorder now! Reorder from your past orders in just one click. Order by Quote. Quote Number. Add quote number from your quote document. Customer Number. Add customer number from your quote document. To remove a quote go to the Cart. View Quote Example. Catalog Number. Looking for a quick way to design experiments? Try the Workflow Configurator. A convenient tool to build experimental workflows and find products to match your needs.

Log Out. Show More. RNeasy Micro Kit Log in to see your account pricing. Column type Plate type. Mini QIAcube. RNeasy Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. Highly reproducible yields for sensitive applications.

High-quality total RNA from fine needle aspirates. A fine needle aspirate was obtained from rabbit liver using a Efficient on-column removal of genomic DNA. Of the "-RT" results, only the 50, cell data points are visible. Significant time savings with QIAcube Kits. High-quality RNA from a variety of samples. Reliable RNA isolation from a single cell.

High-throughput processing in well format The RNeasy 96 system provides a fast and efficient procedure. Supporting data and figures Reliable RNA isolation from a single cell.