Name: Fatty Tissue RNA Purification Kit
Brand: Norgen Biotek Corp.
SKU: 36200
Price: 229.000000 USD
Availability: InStock

Supporting Data

Figure 1. Isolation of High Quality Brain Total RNA including microRNA without the use of Phenol. Norgen's Fatty Tissue RNA Purification Kit isolates high quality total RNA from fatty tissues such as brain without the use of phenol. Total RNA was isolated from equal amounts of brain tissue using Norgen’s Fatty Tissue RNA Purification Kit and a leading competitor’s kit that involves a phenol:chloroform extraction step. The purified RNA was then resolved on a 1.2% formaldehyde-agarose gel. As it can be seen, Norgen not only isolated high and consistent yields of total RNA, but the RNA was also of high quality as evidenced by intactness of the major 28S and 18S rRNA. Importantly, only Norgen's Fatty Tissue RNA Purification Kit was able to recover the small RNA fraction, including the microRNA (red-boxed).

Figure 2. High Yield of a Diversity of RNA Species. Norgen's Fatty Tissue RNA Purification Kit effectively recovers all sizes of RNA from large mRNA to small RNA, including microRNAs. Total RNA was isolated from equal amounts of brain tissue using Norgen's Fatty Tissue RNA Purification Kit and a leading competitor’s kit that involves a phenol:chloroform extraction step. The purified RNA was then used as the template in a RT-qPCR for detecting the beta-Actin gene (Top Panel) and for detecting miR-21 (Bottom Panel). In both graphs the blue lines correspond to Norgen isolated-RNA and the red lines correspond to competitor-isolated RNA. As it can be seen, Norgen's kit isolated higher yields of microRNA, as indicated by the lower Ct values of the blue lines (Top Panel). Also, Norgen's kit successfully isolated similar amount of large RNA to that of the competitor's kit (Bottom Panel) indicating the full diversity of RNA species isolated.

Kit Specifications
Maximum Column Binding Capacity	50 µg
Maximum Column Loading Volume	650 µL
Size of RNA Purified	All sizes, including small RNA (<200 nt)
Maximum Amount of Starting Material: Brain and Adipose Tissues	20 mg
Time to Complete 10 Purifications	50 minutes
Average Yield* Brain (15 mg)	15 µg

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

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Component	Cat. 36200 (25 preps)
Buffer RL	30 mL
Lysis Additive B	1 mL
RNase-Free Water	15 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Mini Spin Columns	50
Collection Tubes	50
Elution Tubes (1.7 mL)	50
Product Insert	1

Documentation

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FAQs

Spin Column

Why do I have poor RNA recovery?

Poor RNA recovery could be due to one or more of the following:

Incomplete lysis of cells or tissue.
Ensure that the appropriate amount of Buffer RL 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 Elution Solution A supplied with this kit be used for maximum RNA recovery.
Ethanol and Buffer RL was not added to the lysate.
Ensure that the appropriate amount of ethanol and Buffer RL is added to the lysate before binding to the column during the RNA purification step.
Ethanol was not added to the Wash Solution A.
Ensure that 90 mL of 96 - 100% ethanol is added to the supplied Wash Solution A prior to use.
Low RNA content in tissue used.
Different tissues 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.
Lysate was not diluted before removal of genomic DNA.
Ensure that 300 µL of RNase-Free Water is added to the lysate prior to genomic DNA removal by a spin column.

I noticed the column was clogged. What causes this?

Column clogging can result from one or a combination of the following factors:

Maximum amount of tissue exceeds kit specifications.
Refer to specifications to determine if the amount of starting material falls within kit specifications.
Centrifuge temperature too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15°C may cause precipitates to form that can cause the columns to clog.

Why is the RNA degrading?

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.
In order 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 protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized.
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 were allowed to thaw prior to RNA isolation.
Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised.

Why is the 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 3 times with the provided Wash Solution A.
Traces of salt from the binding step may remain in the sample if the column is not washed 3 times 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 Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

I noticed genomic DNA contamination. How to prevent/correct this?

Genomic DNA contamination could be because the lysate was diluted too much for removal of genomic by column. Ensure that 315 µL of RNase-Free Water is added to the lysate prior to genomic DNA removal by a spin column.

Can the Fatty Tissue RNA Purification Kit be used to extract RNA from cultured cells containing lipid droplets?

Yes, the Fatty Tissue RNA Purification Kit (#36200) can be used for cells with lipid droplets. Up to 2 million cells per extraction can be processed. Please feel free to contact our Tech support team at support@norgenbiotek.com for help with protocol.

Fatty Tissue RNA Purification Kit