Application Note

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. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, both free circulating[1] and within exosomes[2], as well as within other biofluids such as cerebral spinal fluid[3], urine[4,5] and even breast milk[6]. Circulating miRNAs in plasma/serum represent a potential new approach for diagnostic screening. Indeed, characteristic changes in the serum or plasma miRNA profiles of several cancers[7] or other conditions[8] have identified unique signatures that could be exploited as novel biomarkers in the clinic.

Collecting serum/plasma samples for miRNA analysis:

  1. Collect whole blood into the appropriate commercially available collection tubes.

Serum  – untreated tubes
Plasma – anticoagulant-treated tubes (i.e. EDTA-treated or citrate-treated). Heparinized tubes are not recommended.

  1. Follow the tube manufacturer’s recommendation for collection protocol.
  2. Keep the samples cold and limit the time to analysis. (follow manufacturer’s recommendation for storage times/temps)

Extracting total RNA from serum/plasma samples for miRNA analysis:

We recommend Norgen’s Plasma/Serum RNA Purification Kit because it does not require phenol/chloroform or any protease treatments. RNA can be isolated from either fresh or frozen samples and the kits allow the user to elute into a flexible elution volume ranging from 10 μL to 100 μL.

Select the appropriate kit based on your sample input volume:

  1. Sample Volume- 50-200 µL  – Plasma/Serum RNA Purification Kit #55000
  2. Sample Volume- 0.25-2 mL – Plasma/Serum RNA Purification (Slurry Format) #51000
  3. Sample Volume- 2-5 mL – Plasma/Serum RNA Purification (Slurry Format) #50900

These kits are suitable for the isolation of RNA from serum or plasma prepared from blood collected on either EDTA or citrate treated tubes.

Expected Yields

Typical yields of free circulating and exosomal RNA vary depending on the input sample, as the amount of RNA present in plasma and serum will depend upon the health status of the individual. Normally, the RNA yield from plasma or serum RNA is highly variable (range from 1 to 100ng/mL). Variability can also be observed between samples collected from the same donor at different times during the day.

Quality Control of RNA Samples

You should expect to see lower than normal UV ratios for serum/plasma samples. Please contact us if you are unsure. See this technical bulletin for more specifics on QC of your total RNA sample.

  1. Cheng Y, Tan N, Yang J, Liu X, Cao X, He P, Dong X, Qin S, Zhang C. (2010) A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clinical Science 119, 87-95.
  2. Duelli DM, Hastings ML, Hearn SA, Iyengar R, Kim DJ, Yaddanapudi SC, Pigati L. (2010) Selective release of microRNA species from normal and malignant mammary epithelial cells. PloS ONE 5(10), e13515.
  3. Alexandrov PN1, Dua P, Hill JM, Bhattacharjee S, Zhao Y, Lukiw WJ. (2014) microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF). Int J Biochem Mol Biol 3(4), 365-73.
  4. Taha A Haj-Ahmad, Moemen AK Abdalla, Yousef Haj-Ahmad. (2014) Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients. J Cancer 5(3), 182–191.
  5. Gildea JJ, Carlson JM, Schoeffel CD, Carey RM, Felder RA. (2013) Urinary Exosome miRNome Analysis and its Applications to Salt Sensitivity of Blood Pressure. Clin Biochem 46(12), 1131-4.
  6. Zhou Q, Li M, Wang X, Li Q, Wang T, Zhu Q, Zhou X, Wang X, Gao X, Li X. (2012) Immune-related microRNAs are abundant in breast milk exosomes. Int J Biol Sc 8(1), 118-23.
  7. Ali S, Almhanna K, Chen W, Philip PA, Sarkar FH. (2010) Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer. Am J Transl Res 3(1), 28-47.
  8. Redell JB, Moore AN, Ward Iii NH, Hergenroeder GW, Dash PK. (2010) Human traumatic brain injury alters plasma microRNA levels. J Neurotrauma 27(12), 2147-56.

Identification of novel signature phosphoprotein binding motifs using high density microfluidic PepArray technology MicroRNA Sample Preparation from Biofluid Samples