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Cat. No. / ID: 74124
✓ 24/7 automatic processing of online orders
✓ Knowledgeable and professional Product & Technical Support
✓ Fast and reliable (re)-ordering
RNeasy Protect Mini Kits enable stabilization of RNA in tissue samples, RNA and DNA in sorted or cultured cells, RNA in human saliva samples and RNA in bacterial samples. The RNeasy Protect Mini Kit and Bacteria Mini Kit include RNeasy spin columns for purifying up to 100 µg of high-quality RNA using silica-membrane technology. The RNeasy Protect Cell Mini Kit provides the RNeasy Plus Mini Kit for purification of total RNA. The RNeasy Protect Saliva Mini Kit includes the RNeasy Micro Kit, which purifies and concentrates total RNA using specialized RNeasy MinElute spin columns.
The RNeasy Protect Mini, RNeasy Protect Cell Mini and RNeasy Protect Bacteria Mini Kits can be automated on the QIAcube Connect.
The RNA-stabilizing properties of RNAprotect Tissue Reagent prevent gene induction or down-regulation triggered by sample manipulation allowing you to preserve and analyze the gene expression profile. The RNeasy and RNAprotect bundle enables isolation of high-quality RNA (see figure " Prevention of degradation of mRNA in tissues"). In addition, RNeasy purified RNA from RNAprotect stabilized samples remains intact following storage of tissue samples under a wide variety of conditions (see figure " Stable RNA in tissues"). A range of tissue types can be processed with the RNeasy Protect Mini Kit (see table “Total RNA yields obtained with RNeasy Protect Mini Kit”).
Total RNA yields obtained with RNeasy Protect Mini Kit
|Mouse tissue||Amount (mg)||Average yield* (µg)|
The RNeasy Protect Cell Mini Kit is ideal for gene expression analysis experiments where cells need to be delivered from one lab to another (see table “Higher RNA yields from leukocytes* preserved for 10 days in RNAprotect Cell Reagent”).
Higher RNA yields from leukocytes* preserved for 10 days in RNAprotect Cell Reagent
|Assay result||RNAprotect Cell Reagent||RNAprotect Tissue Reagent|
|RNA yield using RNeasy technology (µg)||10||3.5|
|Real-time RT-PCR of abl (CT value)||25||26|
Cells in RNAprotect Cell Reagent can be stored or transported for up to 1 day at 30ºC, up to 7 days at room temperature (15–25ºC), or up to 4 weeks at 2–8ºC, and can also be stored long-term at –20ºC or –80ºC (see table “Intact RNA from Jurkat cells stored at ambient temperature”, and figures " Effective inhibition of PMA induction" and " More reliable quantification of CD83 transcript") allowing you to preserve and analyze the gene expression profile.
Intact RNA from Jurkat cells stored at ambient temperature
|Cell storage conditions||RIN value of purified RNA*|
|30ºC (1 day)||9.8|
|25ºC (7 days)||9.7|
|2–8ºC (28 days)||10|
|–20ºC (28 days)||9.8|
|–80ºC (28 days)||9.8|
Saliva stabilized with RNAprotect Saliva Reagent can be stored or transported for up to 1 day at 37°C, up to 14 days at room temperature (15–25°C), or up to 4 weeks at 2–8°C, or can be archived at –20°C or –80°C (see figure " Effective stabilization of β-actin transcript") allowing you to preserve and analyze the gene expression profile. Purified RNA from saliva, as an alternative to blood, allows microarray analysis for research into oral and systemic diseases, and gene expression analysis of RNA biomarkers for cancer by real-time RT-PCR (see figures " Minimization of transcript degradation in saliva" and " Stable mRNA levels of cancer biomarkers").
During traditional methods for cell harvesting and RNA isolation, enzymatic degradation of RNA leads to reduction or loss of many transcripts. The reduction is particularly significant in bacterial mRNA molecules, which have very short half lives of only a few minutes. In addition, genes can be induced during handling and processing of bacterial samples, leading to higher expression. Use of RNAprotect Bacteria Reagent overcomes these problems by providing immediate stabilization prior to RNA isolation (see figures " RNAprotect Bacteria Reagent prevents mRNA degradation" and " Accurate gene expression profiles") allowing you to preserve and analyze the gene expression profile. Yield from E. coli (6 x 108 cells) is 100 µg of RNA and the RNA yield from Bacillus subtilis (1 x 108 cells) is 15 µg. RNA purified with the kit is high-quality with A260/A280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5).
RNA purified with RNeasy technology has A260/A280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5). Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.
The RNeasy Protect system – consisting of an RNeasy and RNAprotect bundle – provides a complete RNA protection and isolation solution, from sample harvesting to pure RNA, in one kit. Proven RNeasy silica-membrane technology in spin-column format, combined with the RNA-stabilizing properties of RNAprotect Reagents, allows purification of high-quality, intact RNA. Once a biological sample is harvested, its RNA becomes extremely unstable (see figure " Changes in mRNA levels"). Innovative RNAprotect Reagents immediately stabilize and protect the RNA expression pattern. Samples can be archived without risk of RNA degradation, even after multiple freeze–thaw cycles. Following stabilization, RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification (see figure " RNeasy Mini spin column").
Samples can be stabilized and stored in RNAprotect Tissue Reagent, included in RNeasy Protect Kits. For RNA purification, samples (0.5–30 mg tissue) are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane (binding capacity up to 100 µg RNA). RNA binds, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in 30–100 µl water (see flowchart "RNeasy Protect Mini procedure"). RNA purification can be automated on the QIAcube Connect.
Sorted or cultured cells are mixed with RNAprotect Cell Reagent, which immediately stabilizes the cellular RNA. After RNA stabilization, total RNA is purified using the RNeasy Plus Mini Kit (see flowchart " RNeasy Protect Cell procedure"). The cells in RNAprotect Cell Reagent are centrifuged, and the resulting cell pellet is lysed and homogenized in Buffer RLT Plus. The lysate is then passed through a gDNA Eliminator spin column, which rapidly and effectively removes genomic DNA. Ethanol is added to the lysate, which is then applied to an RNeasy spin column. After centrifugation, total RNA binds to the membrane of the RNeasy spin column. Contaminants are efficiently washed away, and high-quality total RNA is eluted in 30–50 µl of RNase-free water. Purification of DNA, or of RNA and DNA, requires the additional purchase of a QIAGEN DNA purification kit. Details are provided in the RNAprotect Cell Reagent Handbook.
Saliva (200 µl) collected from a donor is immediately mixed with RNAprotect Saliva Reagent to stabilize the RNA in the saliva. Total RNA is purified from stabilized saliva using the RNeasy Micro Kit (see flowchart " RNeasy Protect Saliva procedure"). The saliva sample is first centrifuged, and Buffer RLT is then added to the resulting pellet. After addition of ethanol, the sample is applied to an RNeasy MinElute spin column, where RNA binds to the membrane after centrifugation. Traces of genomic DNA are removed by DNase digestion, and contaminants are washed away in several wash steps. Highly pure RNA is then eluted using RNase-free water in a volume of just 14 µl. RNAprotect Saliva Reagent also stabilizes DNA. Protocols for purification of DNA, or RNA and DNA, from stabilized saliva are available.
Two volumes of RNAprotect Bacteria Reagent are added directly to 1 volume of bacterial culture (≤7.5 x 108 bacteria) prior to RNA isolation, providing immediate stabilization of RNA (see flowchart " RNAprotect Bacteria Reagent procedure"). The stabilization allows time for efficient bacterial lysis using a choice of protocols: enzymatic lysis, mechanical disruption, or a combination of both methods. We recommend the TissueLyser II for efficient mechanical disruption. Ethanol is then added to the lysate to provide ideal binding conditions. The lysate is loaded onto the RNeasy silica membrane (binding capacity 100 µg RNA). Following RNA binding, all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in 30–100 µl of water. The RNeasy Protect Bacteria Mini Kit can be automated on the QIAcube Connect.
Amounts of RNA isolated from bacteria can vary due to species and growth conditions. The RNeasy Protect Bacteria Mini Kit is suitable for use with a wide range of bacterial species, both Gram positive (e.g.,Staphylococcus aureus and Mycobacterium avium) and Gram negative (e.g.,Escherichia coli and Salmonella typhimurium). Bacteria grown in either minimal or complex medium can be used. Since the RNeasy procedure enriches for RNA species >200 nucleotides, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs. RNeasy Protect Bacteria Kits provide the highest-quality RNA with minimum copurification of DNA. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using the RNase-Free DNase Set for convenient on-column DNase treatment during the RNeasy procedure.
RNA purified with RNeasy technology is ideal for use in all applications. Downstream applications include:
|Features||RNeasy Protect Mini Kit||RNeasy Protect Cell Mini Kit||RNeasy Protect Saliva Mini Kit||RNeasy Protect Bacteria Mini Kit|
|Applications||RNA-seq, PCR, qPCR, real-time RT-PCR, microarray||RNA-seq, PCR, qPCR, real-time RT-PCR, microarray||RNA-seq, PCR, qPCR, real-time RT-PCR, microarray||RNA-seq, PCR, qPCR, real-time RT-PCR, microarray|
|Elution volume||30–100 µl||30–50 µl||8–14 µl||30–100 µl|
|Format||Spin column||Spin column||Spin column||Spin column|
|Main sample type||Tissue samples||Cultured cells||Saliva samples||Bacteria|
|Processing||Manual (centrifugation)||Manual (centrifugation)||Manual (centrifugation)||Manual (centrifugation)|
Purification of total RNA, miRNA,
poly A+ mRNA, DNA or protein
|Sample amount||0.5–30 mg||Max. 107 cells||200 µl||15–100 µg|
|Technology||Silica technology||Silica technology||Silica technology||Silica technology|
|Time per run or per prep||–||–||–||20 minutes|
|Yield||10–45 µg||Varies||Varies||8–70 µg|
Yes, we recommend to use the RNeasy Protect Cell Mini Kit which contains RNAprotect Cell Reagent, the first RNA stabilization reagent for cultured cells in the market optimized for a broad range of cell types and cell culture formats.
Due to the complex nature of cartilage we would recommend to use the RNeasy Lipid Tissue Kit. Follow the standard tissue protocol in the RNeasy Lipid Tissue Kit Handbook.
To help you choose the correct RNeasy kit for the isolation of total RNA from different types of tissue, please refer to our Kit Selection Guide.
No, the RNeasy Protect Saliva Mini Kit does not contain a saliva collection device. Any vessel, e.g. 50 ml polypropylene tubes, can be used.
Yes, RNAprotect Bacteria Reagent and the RNeasy Mini Kit are compatible. You can also purchase QIAGEN's RNeasy Protect Bacteria Kits, which will contain RNAprotect Bacteria Reagent and RNeasy chemistry. Protocols for isolating RNA from bacteria after using the Reagent for bacterial RNA stabilization can be found in the RNAprotect Bacteria Reagent Handbook.
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. For single tube format RNA purification, the RNeasy Plus Universal Kit is also available.
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. no. 79216)
Note: note that ß-mercaptoethanol should be added to Buffer RLT before use to effectively inactivate RNAses in the lysate (10 µl ß-Mercaptoethanol per 1 ml Buffer RLT).
Avoid RNA degradation due to improper sample storage and handling prior to the extraction procedure with RNeasy Kits. 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. Samples can be immediately flash frozen in liquid nitrogen and stored at −90 to −65°C. as soon as they are harvested or excised. 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. Samples can also be stored at −90 to −65°C. in lysis buffer (Buffer RLT) after disruption and homogenization. Frozen samples are stable for months.
For optimal RNA yields with RNeasy Kits it is crucial to:
Please review the instructions in the relevant RNeasy Handbook carefully for best results.
The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing-agarose gel electrophoresis, the Agilent 2100 bioanalyzer, or the QIAxcel Advanced System with the QIAxcel RNA QC Kit v2.0.
The respective ribosomal species should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should be present with an intensity approximately twice that of the 18S RNA band. 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.
Size of ribosomal RNAs from various sources
Purity of RNA isolated with RNeasy Kits can be evaluated by determining the ratio of absorbance readings at 260 nm and 280 nm (A260/A280). This ratio provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV range, such as protein.
Note that the A260/A280 ratio is influenced considerably by pH. As water is unbuffered, the pH and the resulting 260/280 ratio can vary greatly. For an accurate determination of purity, we recommend measuring the 260/280 absorbance in 10 mM Tris-Cl, pH 7.5. Be sure to calibrate the spectrophotometer with the same solution. Pure RNA has an A260/A280 ratio of 1.9-2.1. However, values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris, pH 7.5) with some spectrophotometers.
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. 1997 Mar;22(3):474-6, 478-81.
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. For details please refer to the chapter "A Guide to Analytical Gels" in the QIAGEN Bench Guide.
Some banding pattern may remain due to the presence of mRNA transcripts of different lengths specific for the respective cell or tissue type.
Yes, RNAprotect Cell Reagent of the RNeasy Protect Cell Mini Kit can be purchased separately in a bulk size of 250 ml for processing large cell culture volumes.
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. Incomplete homogenization results in genomic DNA contamination, and inefficient binding of RNA to the RNeasy membrane resulting in reduced yields.
Yes. Even though buffer RDD in the RNase-Free DNase Set is optimized for on-column DNase digestion, the buffer is also well-suited for efficient DNase digestion in solution. 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.
A protocol for in-solution DNase digestion using the RNase-Free DNase Set can be found in Appendix C of the RNeasy MinElute Cleanup Handbook. For subsequent RNA Cleanup, use either the RNeasy MinElute Cleanup Kit, or follow the instructions for RNA Cleanup in the RNeasy Mini Handbook.
In comparison to Buffer RLT of, e.g., the RNeasy Mini Kit, Buffer RLT Plus of the RNeasy Plus Mini Kit and RNeasy Plus 96 Kit also contains a proprietary blend of detergents that aid in the binding of genomic DNA to the gDNA Eliminator Mini Spin Columns, or to the gDNA Eliminator 96 plate respectively.
We do not recommend to store previously isolated RNA in RNAprotect Tissue Reagent, since it will be very difficult to recover isolated RNA from it. Purified RNA can be stored at −30 to −15°C or −90 to −65°C in sterile RNase-free water. Under these conditions, we found no detectable RNA degradation after 1 year of storage.
No, RNAprotect Saliva Reagent is provided in the RNeasy Protect Saliva Mini Kit only, and is not available separately.
Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.
Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).
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. Use of 3-5% ß-mercaptoethanol in Buffer RLT instead of 1% may also improve the results.
Our RNeasy buffers are subjected to stringent quality-control tests to ensure that they are indeed RNase-free. Buffer RPE concentrate and RNase-free water are tested for absence of RNases by incubating 4 µg of total HeLa-RNA in these solutions for 3 hours at 37°C, followed by monitoring RNA integrity via denaturing agarose gel electrophoresis and ethidium bromide staining.
Buffer RLT and Buffer RW1 are inherently RNase-free, since the buffers themselves inactivate RNases during the RNeasy procedure.
No. The addition of beta-mercaptoethanol to lysis buffer RLT used in the RNeasy Kits is sufficient to inactivate any RNases in your sample.
Yes, but you have to make sure that the RNAprotect Bacteria Reagent is diluted in PBS or water prior to use (2 volumes reagent : 1 volume PBS or water) in order to achieve its optimal working concentration. The diluted reagent is directly added to the bacteria growing on the solid substrate, and reagent and bacteria are mixed thoroughly. The reagent is not recommended for stab cultures, since sufficient access of the reagent to all bacteria for immediate RNA stabilization cannot be guaranteed.
The efficiency of downstream applications depends strongly on the purity of the RNA sample used. Pure RNA should yield an A260/A230 ratio of around 2 or slightly above; however, there is no consensus on the acceptable lower limit of this ratio. Possible candidates that can increase the A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general). In our experience, the increased absorbance at 230 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.
Please find an article discussing the effect of low 260/230 ratios in RNA preparations on downstream applications on page 7 of QIAGEN Newsletter March 15, 2010 . In summary, we found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications.
For RNA isolated on the BioRobot EZ1 and BioRobot M48:
The RNA can be directly applied to the Agilent Bioanalyzer, since it is being denatured in the final protocol steps of these isolation procedures.
For RNA prepared with all other QIAGEN RNA Isolation Products:
We recommend to denature the samples in a water bath for 2 min at 70°C, and then place them directly on ice prior to loading them onto the Agilent Bioanalyzer.
If the cells in RNAprotect Tissue Reagent cannot be collected by centrifugation, please try one of the following suggestions:
1) Add 600 µl Buffer RLT to a maximum of 200 µl sample volume, and proceed with step 3 of the "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells" in the RNeasy Mini Handbook. Load the lysate onto the column in successive aliquots in step 5 of the protocol.
2) If cells are floating on the surface of the RNAprotect Tissue Reagent, try removing the reagent by pipetting from underneath. Leave behind approximately 100 ul of RNAprotect Tissue Reagent, and add 350 ul Buffer RLT before proceeding with the protocol "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells". For every 100 ul of cells in RNAprotect Tissue Reagent, use 250 ul of 96-100% ethanol instead of the 70% ethanol listed in step 4 of the standard protocol.
3) Dilute the sample 10x by adding cold PBS. Pellet cells by centrifugation. Caution: Cells might lyse.
4) Try to pipette the floating cells off the surface.
Note that the above steps are suggestions, rather than official protocol recommendations. Please try a "pilot" run on a test sample first.
For RNA stabilization of cells, we recommend using the RNAprotect Cell Reagent.
Tissues preserved in RNAprotect Tissue Reagent can be stored as long as listed below:
|37 °C||1 day|
|room temp.||1 week|
|2-8 °C||1 month|
|-30 to -15 °C||archival|
|-90 to -65 °C||archival|
To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:
In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.
Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.
The A260/ A280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results in a lower A260/ A280 ratio and a reduced sensitivity to protein contamination*.
For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution. Please see the Appendix sections in the RNeasy handbooks for additional information.
* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.
Always dispose of potentially biohazardous solutions according to your institution’s waste-disposal guidelines. 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.
Please access our Material Safety Data Sheets (MSDS) online for detailed information on the reagents for each respective kit.
The RNeasy Protect Saliva Mini Kit is based on RNeasy MinElute technology, which allows elution volumes as low as 10-14 µl, thereby concentrating saliva RNA.
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.
Beta-mercaptoethanol (ß-ME) is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality. 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.
The exact composition of Buffer RW1 is confidential. Buffer RW1 is a proprietary component of RNeasy Kits. 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., that are non-specifically bound to the silica membrane. At the same time, RNA molecules larger than 200 bases remain bound to the column.
Note: Buffer RW1 should not be used for isolation of small RNAs, for example, microRNAs or fragmented RNA from formalin-fixed tissues, as these smaller fragments will be washed away. Buffer RWT should be used instead.
Yes. The RNeasy Mini Kit and RNeasy 96 Kit have been used successfully to isolate RNA from fewer than 100 cells. We recommend adding 20 ng of carrier RNA to the cell lysate before loading it onto the RNeasy membrane. Please note that the carrier RNA will co-purify with cellular RNA. However, the small amounts of poly-A RNA used as carrier RNA do not interfere with subsequent RT-PCR, even when oligo-dT is used for reverse transcription. 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.
Alternatively, the RNeasy Micro Kit can be used for isolating RNA from up to 5x 105 cells. The RNeasy Micro procedure uses a novel technology to purify RNA from small amounts of tissues or cells (as little as 1 cell).
The concentration of RNA isolated with RNeasy Kits can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Absorbance readings should be greater than 0.15 to ensure significance. An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per ml (A260 = 1 = 40 µg/ml). 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:
RNA concentration: 460 µg/ml
RNA yield: 46 µg
For additional information on RNA quantitation and handling, see the Appendix section in the RNeasy Mini Handbook.