Cytoplasmic and Nuclear RNA Purification Kit
For the convenient purification of cytoplasmic and nuclear RNA from cultured cells and tissues
For research use only and NOT intended for in vitro diagnostics.
Cytoplasmic and Nuclear RNA Purification Kit
For the convenient purification of cytoplasmic and nuclear RNA from cultured cells and tissues
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Features and Benefits
- Excellent separation and purification of cytoplasmic and nuclear RNA
- Convenient and fast spin column format
- High quality and yield of RNA
- Isolate full diversity of RNA (including microRNA) without phenol
- Purified RNA is ready for any application including RT-PCR, qRT-PCR, RNA-Seq, arrays and more
- Cytoplasmic RNA is free of DNA and ready for direct use in RT-PCR/qRT-PCR
- Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix
This kit provides a rapid method for the isolation and purification of both cytoplasmic and nuclear RNA from cultured animal cells and small tissue samples. The kit can be used to isolate all sizes of RNA from the cytoplasmic and nuclear RNA fractions, including all small RNA species without any requirement for phenol. Included in the kit are sufficient reagents to perform either 50 cytoplasmic RNA preparations or 25 cytoplasmic and 25 nuclear RNA preparations. Ten samples can be processed in approximately 45 minutes. This kit is also available in a 100 prep size.
Background
In certain circumstances it is desirable to be able to isolate fractionated RNA as opposed to total RNA. For example, it may be preferable to isolate only mature, cytoplasmic RNA for some studies on expression profiling. Alternatively it may be desirable to isolate nuclear RNA in order to investigate and study pre-processed (non-spliced) RNA. Furthermore, this kit can be used to isolate RNA for downstream applications where it is necessary to avoid DNA contamination, since the cytoplasmic fraction has been shown to be free of all traces of genomic DNA.
Details
Supporting Data
Kit Specifications
|
|
Maximum Column Binding Capacity
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Up to 50 µg RNA
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Maximum Column Loading Volume
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650 µL
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Size of RNA Purified
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All sizes, including small RNA (< 200 nt)
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Time to Complete 10 Purifications |
45 minutes
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RNA Yield
HeLa (1 x 106) - Cytoplasmic RNA HeLa (1 x 106) - Nuclear RNA |
15 µg ≤ 3.5 µg |
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. 21000 (50 preps) | Cat. 37400 (100 preps) |
---|---|---|
Lysis Buffer J | 20 mL | 2 x 20 mL |
Buffer SK | 40 mL | 2 x 40 mL |
Wash Solution A | 38 mL | 2 x 38 mL |
Elution Buffer E | 6 mL | 2 x 6 mL |
Mini Spin Columns | 50 | 100 |
Collection Tubes | 50 | 100 |
Elution Tubes (1.7 mL) | 50 | 100 |
Product Insert | 1 | 1 |
Documentation
FAQs
Spin Column
Poor RNA recovery could be due to one or more of the following:
- Insufficient solubilization of cells or tissue.
Ensure that the appropriate amount of Lysis Buffer J 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 Buffer E 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 90 mL of 96-100% ethanol is added to the supplied Wash Solution A prior to use.
- 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.
Column clogging can result from one or a combination of the following factors:
- Insufficient solubilization of cells or tissues.
Ensure that the appropriate amount of Lysis Buffer J was used for the amount of cells or tissue.
- 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.
- High amounts of genomic DNA present in sample.
The nuclear lysate fraction may be passed through a 25 gauge needle attached to a syringe 5-10 times in order to shear the genomic DNA prior to loading onto the column.
- Centrifuge temperature is too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15℃ may cause precipitates to form that can cause the columns to clog.
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 Animal Tissue 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℃ for a few days. It is recommended that samples be stored at –70℃ 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 in order to ensure that the integrity of the RNA is not compromised.
- Tissue samples were frozen improperly.
Samples should be flash-frozen in liquid nitrogen and transferred immediately to a -70℃ freezer for long-term storage.
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.
Genomic DNA contamination could be because traces of nuclear pellet remained in cytoplasmic fraction. Ensure that a solid pellet is formed at the end of the Cell Fraction Preparation step, and that none of the pellet is removed when the supernatant is transferred to another tube.
Please refer to supporting data Fig 4 where we have shown a qPCR for U2 snRNA which is amplified only in nuclear fraction, and also a qPCR for S14 RNA which is amplified in cytoplasmic fraction.
Citations
Title | Long non-coding RNA MSTRG.5970.28 regulates proliferation and apoptosis of goose follicle granulosa cells via the miR-133a-3p/ANOS1 pathway |
Citation | Poultry Science 2023. |
Authors | Xiaoyu Zhao, Haiying Li, Xingyong Chen, Yingping Wu, Ling Wang, Jiahui Li, Xiaoyu Zhao, Haiying Li, Xingyong Chen, Yingping Wu, Ling Wang, Jiahui Li, |
Title | LncRNA RP11-620J15.3 promotes HCC cell proliferation and metastasis by targeting miR-326/GPI to enhance glycolysis |
Citation | Biology Direct volume 2023. |
Authors | Chuanjiang Liu, Kequan Xu, Jiayin Liu, Chao He, Pan Liu, Qiang Fu, Hongwei Zhang & Tao Qin |
Title | Long non-coding RNA LTCONS8875 regulates innate immunity by up-regulating IRAK4 in Miichthys miiuy (miiuy croaker) |
Citation | Developmental & Comparative Immunology 2023. |
Authors | Quiang Luo, Xing Lv, Liyuan Yang, Weiwei Zheng, TIanjun Xu, Yuena Sun |
Title | Long noncoding RNA LTCONS4500 promotes antibacterial immune responses via targeting miR-3570-5p in teleost fish Miichthys miiuy |
Citation | Developmental & Comparative Immunology 2023. |
Authors | Qing Chu, Jingyao Yu, Jiale Zhou |
Title | Hsa_circ_0022383 promote non-small cell lung cancer tumorigenesis through regulating the miR-495-3p/KPNA2 axis |
Citation | Cancer Cell International 2023. |
Authors | Xiaofang Xu, Binbin Song, Qiuliang Zhang, Weibo Qi & Yufen Xu |
Title | Long noncoding RNA long intergenic non-protein-coding RNA 173 contributes to nasopharyngeal carcinoma progression by regulating microRNA-765/Gremlin 1 pathway |
Citation | Human & Experimental Toxicology 2023. |
Authors | Dan Wang and Heng Jiang |
Title | Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B |
Citation | The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology 2023. |
Authors | Xiaofan Xia, Jinxiu Fan, Zhongjie Fan |
Title | Long noncoding RNA LINC01088 inhibits esophageal squamous cell carcinoma progression by targeting the NPM1-HDM2-p53 axis |
Citation | Acta Biochimica et Biophysica Sinica 2023. |
Authors | Fan Liang, Qiuli Luo, Haibo Han, Jianzhi Zhang, Yue Yang , and Jinfeng Chen |
Title | hsa_circRNA_BECN1 acts as a ceRNA to promote polycystic ovary syndrome progression by sponging the miR-619-5p/Rab5b axis Get access Arrow |
Citation | Molecular Human Reproduction 2023. |
Authors | Hairui Fan, Dongjie Zhou, Xiaomei Zhang, Min Jiang, Xiang Kong, Tongmin Xue, Lingling Gao, Dan Lu, Chenyue Tao, Liping Wang |
Title | LincRNA00612 inhibits apoptosis and inflammation in LPS-induced BEAS-2B cells via enhancing interaction between p-STAT3 and A2M promoter |
Citation | PeerJ 2023. |
Authors | Xinru Xiao?, Wei Cai?, Ziqi Ding, Zhengdao Mao, Yujia Shi, Qian Zhang |