RNA/DNA Purification Kits

Features and Benefits

Sequentially isolate and purify total RNA and DNA from a single sample
Two column system: one for DNA and one for RNA
The RNA column is for the purification of total RNA including microRNA
No need to split the lysate, or to use phenol or precipitation methods
Rapid and efficient spin column procedure - it takes only 30 minutes
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a rapid method for the isolation and purification of total RNA and DNA sequentially from a single sample of cultured animal cells and tissues, blood, bacteria, yeast, or fungi. The lysate is passed over two columns: 1) a DNA column and 2) an RNA column. Total RNA of all sizes is purified, including microRNA. Both DNA and RNA are of the highest purity and yield.

These kits are ideal for researchers who are interested in studying the genome and transcriptome of a single sample, such as for studies of microRNA profiling, gene expression including gene silencing experiments or mRNA knockdowns, studies involving biomarker discovery, and for characterization of cultured cell lines. Norgen’s RNA/DNA Purification Kits are especially useful for researchers who are isolating macromolecules from precious, difficult to obtain or small samples such as biopsy materials or single foci from cell cultures, as they eliminate the need to fractionate the sample. Furthermore, analysis will be more reliable since the RNA and DNA are derived from the same sample, thereby eliminating inconsistent results. The purified macromolecules are of the highest purity and can be used in a number of different downstream applications

RNA/DNA Purification Kit (Spin Column)

Maximum column binding capacity of 50 μg for RNA and 20 μg for DNA.

RNA/DNA Purification Micro Kit (Micro)

The purified RNA and DNA fractions can be eluted in as little as 20 μL. Ideal for cell number inputs of 500,000 and as little as 5 cells. Maximum column binding capacity of 35 μg for RNA and 10 μg for DNA.

Details

Supporting Data

Figure 1. Recovery of True Total RNA including microRNA from Hamster Heart. Panel A is a 1X MOPS 1% agarose gel showing the RNA that was isolated from 2 different samples of 10 mg hamster heart using Norgen's RNA/DNA Purification Kit. Norgen's RNA/DNA Purification Kit isolated large RNA (represented by 28S and 18S rRNA) with high integrity. Moreover, it provided the added benefit of recovering small RNA without any additional protocol. Panel B is a result from a bioanalyzer resolution of the eluted RNA. Similar to the agarose gel, Norgen's RNA/DNA Purification Kit showed the added benefit of recovering small RNA. The effectiveness of small RNA recovery was also demonstrated by gene-specific RT-qPCR. One microgram of RNA was used in RT-qPCR reactions for human S15 (for Large RNA) and miR-21 (for microRNA) genes. The RNA isolated by Norgen’s RNA/DNA Purification Kit showed detection of both small RNA (Panel C) and the large RNA (Panel D).

Figure 2. Recovery of Intact, High Quality Genomic DNA from Hamster Heart. Panel A is a 1% agarose gel showing the gDNA isolated from the same hamster heart samples above using Norgen's RNA/DNA Purification Kit. Lane M is Norgen's HighRanger 1 kb DNA Ladder and the sample lanes contain 10 µL of each of the 100 µL elutions. The gel showed high quality, and intact genomic DNA. Panel B is the result of qPCR amplification of 25 ng of eluted genomic DNA using 5S rRNA-specific primers. Genomic DNA isolated using Norgen's RNA/DNA Purification Kit is of high quality with effective qPCR amplification.

Figure 3. Recovery of True Total RNA including microRNA from Hamster Liver. Panel A is a 1X MOPS 1% agarose gel showing 3 µL of 20 µL eluted RNA that was isolated from 2 different samples of 5 mg hamster liver using Norgen's RNA/DNA Purification Micro Kit. Norgen’s RNA/DNA Purification Micro Kit isolated large RNA (represented by 28S and 18S rRNA) with high integrity. Moreover, it provided the added benefit of recovering small RNA without any additional protocol. Panel B is a result from a bioanalyzer resolution of the eluted RNA. Similar to the agarose gel, Norgen's RNA/DNA Purification Micro Kit showed the added benefit of recovering small RNA while isolating very high quality RNA. The effectiveness in small RNA recovery was also demonstrated by gene-specific RT-qPCR. One microgram of RNA was used in RT-qPCR reactions for beta-Actin (for Large RNA) and miR-21 (for microRNA) genes. The RNA isolated by Norgen’s RNA/DNA Purification Micro Kit showed detection of both small RNA (Panel C) and the large RNA (Panel D).

Figure 4. Recovery of Intact, High Quality Genomic DNA from Hamster Liver. Panel A is a 1% agarose gel showing the gDNA isolated from the same hamster liver samples using Norgen’s RNA/DNA Purification Micro Kit. Lane M is Norgen's HighRanger 1 kb DNA Ladder and the sample lanes contain 3 µL of each of the 20 µL elutions. The gel showed high quality, and intact genomic DNA. Panel B is the result of qPCR amplification of 25 ng of eluted genomic DNA using 5S rRNA-specific primers. Genomic DNA isolated using Norgen’s RNA/DNA Purification Micro Kit is of high quality with effective qPCR amplification.

Kit Specifications
Maximum Column Binding Capacity	50 μg for RNA 20 μg for DNA
Maximum Column Loading Volume	650 μL
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Maximum Amount of Starting Material: Animal Cells Animal Tissues Blood Bacteria Yeast Fungi Plant Tissues	5 x 10⁶ cells 25 mg (for most tissues)** 200 μL 1 x 10⁹ cells 1 x 10⁸ cells 50 mg 50 mg
Time to Complete 10 Purifications	30 minutes
Average Yield*: HEK 293 Cells (1 x 10⁶ cells) HEK 293 Cells (1 x 10⁶ cells) Liver (15 mg) Liver (15 mg)	10-15 μg RNA 2-4 μg DNA 30-35 μg RNA 4-6 μg DNA

*Average Yield will vary depending upon a number of factors including species, growth conditions used, and development stage.

**Tissue inputs of up to 40 mg may be used, however for inputs greater than the recommended 25 mg, cross-contamination of the RNA and DNA fractions is possible.

Storage Conditions and Product Stability
Store Proteinase K at -20°C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Component	Cat. 48700 (50 preps)	Cat. 50300 (50 preps)
Buffer SKP	40 mL	40 mL
Wash Solution A	2 x 38 mL 1 x 18 mL	2 x 38 mL 1 x 18 mL
Elution Solution A	6 mL	6 mL
Elution Buffer F	15 mL	6 mL
RNase-Free Water	40 mL	40 mL
Proteinase K	2 x 12 mg	2 x 12 mg
gDNA Purification Columns	50	-
gDNA Purification Micro Columns	-	50
RNA Purification Columns	50	-
RNA Purification Micro Columns	-	50
Collection Tubes	100	100
Elution Tubes (1.7 mL)	100	100
Product Insert	1	1

FAQs

Spin Column, Micro

Why do I have poor DNA recovery?

Poor DNA recovery may be caused by one or more of the following:

Incomplete lysis of cells or tissue. Ensure that the appropriate amount of Buffer SKP was used for the amount of cells or tissue.
Column has become clogged. Do not exceed the recommended amounts of starting materials. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See FAQ related to “Clogged Column” below.
An alternative elution solution was used. It is recommended that the RNA Elution Buffer supplied with this kit be used for maximum RNA recovery.
Ethanol was not added to the lysate. Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
Ethanol was not added to the Wash Solution A. Ensure that 96 – 100 % ethanol is added to the supplied Wash Solution prior to use.
Low RNA content in cells or tissues used. Different tissues and cells have different RNA contents, and thus the expected yield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material.
Cell Culture: Cell monolayer was not washed with PBS. Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells.
Yeast: Lyticase was not added to the Resuspension Buffer. Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer.
Bacteria and Yeast: All traces of media not removed. Ensure that all media is removed prior to the addition of the Buffer SKP through aspiration.

I noticed the column was clogged. What causes this?

Column clogging may occur due to:

Maximum number of cells or amount of tissue exceeds kit specifications. Refer to specifications to determine if the amount of starting material falls within kit specifications.
Centrifuge temperature is too low. Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20°C may cause precipitates to form that can cause the columns to clog.

Why is the eluted RNA degraded?

Eluted RNA can be degraded due to the following factors:

RNase contamination. RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of this user guide.
Procedure not performed quickly enough. To maintain the integrity of the RNA, it is important that the procedure be performed quickly. This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol, as RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized. Also, after the DNA binding step, the flowthrough should be kept on ice or –20°C if the RNA purification step is not carried out immediately.
Improper storage of the purified RNA. For short-term storage, RNA samples may be stored at –20°C for a few days. It is recommended that samples be stored at –70°C for longer-term storage.
Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation. Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle to ensure that the integrity of the RNA is not compromised.
Lysozyme or lyticase used may not be RNase-free. Ensure that the lysozyme and lyticase being used with this kit are RNase-free to prevent possible problems with RNA degradation.
Tissue samples were frozen improperly. Samples should be flash-frozen in liquid nitrogen and transferred immediately to a –70°C freezer for long-term storage.

Why is the purified RNA not performing well in downstream applications?

If the RNA does not perform well in downstream applications, it may be due to one or more of the following:

RNA was not washed twice with the provided Wash Solution A. Traces of salt from the binding step may remain in the sample if the column is not washed twice with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.
Ethanol carryover. Ensure that the dry spin under the RNA Wash procedure is performed to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

Citations

Title	Phenotypic investigations of the depletion of EngA in Escherchia coli are consistent with a role in ribosome biogenesis
Citation	FEMS Microbiology Letters 2014.
Authors	A Bharat, ED Brown