miRNA Sample Preparation from Biofluids

Views:
 
     
 

Presentation Description

The study of basic microRNA (miRNA) function has been performed mainly through collection of tissues by invasive methods. However, in a clinical setting, more convenient and non-invasive methods are required, such as collection of peripheral blood or other bodily fluids. For those of you currently collecting sample from patients or working on development of a clinical test, we have put together some recommendations for sample collection and preservation and for RNA extraction methods from bodily fluids. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, both free circulating and within exosomes, as well as within other biofluids such as cerebral spinal fluid, urine and even breast milk6. Circulating miRNAs in biofluids represent a potential new approach for diagnostic screening. Indeed, characteristic changes in the serum or plasma miRNA profiles of several cancers or other conditions have identified unique signatures that could be exploited as novel biomarkers in the clinic.

Comments

Presentation Transcript

PowerPoint Presentation:

microRNA Sample Preparation from Biofluids

PowerPoint Presentation:

2008 Serum - Chen X et al.(2008) Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006. Plasma - Mitchell PS et al.(2008) Circulating microRNAs as stable blood-based markers for cancer detection. PNAS USA 105:10513–10518. Exosomes - Taylor DD, Gercel -Taylor C. (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 116(1):153. Urine - Melkonyan HS et al. (2008) Transrenal nucleic acids: from proof of principle to clinical tests. Ann N Y Acad Sci. 1137:73-81. Cerebrospinal fluid - Cogswell JP et al. (2008) Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways. J Alzheimers Dis 14(1):27-41.   2009 Saliva - Park NJ et al. (2009) Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin Cancer Res 15(17):5473-7  2010 Cows milk - Hata T et al. (2010) Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs. Biochem Biophys Res Commun 396(2): 528-33. Breast milk - Kosaka N, Izumi H, Sekine K, Ochiya T. (2010) microRNA as a new immune-regulatory agent in breast milk. Silence. 1(1):7 .

PowerPoint Presentation:

Duelli DM et al. (2010) Selective release of microRNA species from normal and malignant mammary epithelial cells. PloS ONE 5(10), e13515. Breast Cancer

PowerPoint Presentation:

Haj-Ahmad TA et al. (2014) Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients. J Cancer 5(3), 182–191. Prostate Cancer

PowerPoint Presentation:

Ali S et al. (2010) Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer. Am J Transl Res 3(1): 28-47. Pancreatic Cancer

PowerPoint Presentation:

Traumatic Brain Injury Redell JB et al. (2010) Human traumatic brain injury alters plasma microRNA levels. J Neurotrauma 27(12): 2147-56.

PowerPoint Presentation:

Whole Blood Serum Plasma Urine Cerebro -spinal Fluid Saliva Breast Milk Collection Tube Norgen's Blood RNA Preservative Tubes or Tempus™ Blood RNA Tubes Untreated tubes Anticoagulant-treated tubes (EDTA-treated or citrate-treated). Norgen’s Urine Preservation Tube Untreated tubes Untreated tubes Untreated tubes Collection Notes Follow the tube manufacturer’s recommendation for collection protocol. Keep the samples cold and limit the time to analysis. First void (mid-stream) urine is highly recommended. Centrifuge prior to aliquoting /freezing to remove cell contamination and/or fat globules.

PowerPoint Presentation:

Whole Blood Serum Plasma Urine Cerebro -spinal Fluid Saliva Breast Milk Collection Tube Norgen's Blood RNA Preservative Tubes or Tempus™ Blood RNA Tubes Untreated tubes Anticoagulant-treated tubes (EDTA-treated or citrate-treated). Norgen’s Urine Preservation Tube Untreated tubes Untreated tubes Untreated tubes Collection Notes Follow the tube manufacturer’s recommendation for collection protocol. Keep the samples cold and limit the time to analysis. First void (mid-stream) urine is highly recommended. Centrifuge prior to aliquoting /freezing to remove cell contamination and/or fat globules.

PowerPoint Presentation:

Whole Blood Serum Plasma Urine Cerebro -spinal Fluid Saliva Breast Milk Collection Tube Norgen's Blood RNA Preservative Tubes or Tempus™ Blood RNA Tubes Untreated tubes Anticoagulant-treated tubes (EDTA-treated or citrate-treated). Norgen’s Urine Preservation Tube Untreated tubes Untreated tubes Untreated tubes Collection Notes Follow the tube manufacturer’s recommendation for collection protocol. Keep the samples cold and limit the time to analysis. First void (mid-stream) urine is highly recommended. Centrifuge prior to aliquoting /freezing to remove cell contamination and/or fat globules.

PowerPoint Presentation:

Whole Blood Serum Plasma Urine Cerebro -spinal Fluid Saliva Breast Milk Collection Tube Norgen's Blood RNA Preservative Tubes or Tempus™ Blood RNA Tubes Untreated tubes Anticoagulant-treated tubes (EDTA-treated or citrate-treated). Norgen’s Urine Preservation Tube Untreated tubes Untreated tubes Untreated tubes Collection Notes Follow the tube manufacturer’s recommendation for collection protocol. Keep the samples cold and limit the time to analysis. First void (mid-stream) urine is highly recommended. Centrifuge prior to aliquoting /freezing to remove cell contamination and/or fat globules.

PowerPoint Presentation:

Norgen Biotek – Total RNA Purification Kit Does not require phenol/chloroform Does not require protease treatment Isolate RNA from fresh or frozen samples Elute in 50 µl to 5 ml volumes

PowerPoint Presentation:

Norgen Biotek – Total RNA Purification Kit 50 – 200 µl 0.25 – 2.0 ml 2.0 – 5.0 ml

PowerPoint Presentation:

Urine Total RNA Purification Kit Urine Exosome RNA Purification Kit

PowerPoint Presentation:

Very low yield of small RNA <200 nt (including microRNA) 1.5% Agarose Gel BioAnalyzer Gel Image Urea-PAGE Gel

PowerPoint Presentation:

Typical UV Ratios 260 nm 260 nm 230 nm 280 nm 1.0 1.8

PowerPoint Presentation:

LC Sciences, LLC 2575 West Bellfort Street, #270 Houston, TX 77054 United States www.lcsciences.com Questions or feedback: 713-664-7087 info@lcsciences.com Thank You!

authorStream Live Help