Labome reviews formal articles for information about instruments and reagents. The following is a summary of microRNA research reagents based on this set of microRNA articles. The inclusion of articles should be considered as random, with regards to reagent types, research methods and suppliers, and thus provide a snapshot (albeit an incomplete list) of the microRNA research reagents.

Most of the articles examine the roles of specific microRNAs in normal or pathological processes, such as the regulation of Pax3 mRNA by miR206 [1], m7G methylation in let-7 [2], the involvement of miR156 system in plant flowering process [3], and miR-92a, miR-454, and miR-320b in neural development [4].

Other articles discuss microRNA biogenesis, global regulation or other processes related to microRNA, cancer cell identification [5], transgenerational epigenetic instability [6], and artificial microRNA [7]. One publication involves piRNAs [8].

Yan X et al transduced LAPC4 cells with System Biosciences pre-mir-34a lentiviral particles (cat# PMIRH34aPA-1) and scramble non-targeting pre-miRNA (cat# PMIRH000PA-1) to study the effect of mir-34a on tumor growth [9].

MicroRNA mimics are small double-stranded RNA molecules with modifications that can be used to either transfect into cultured cells or animals and have similar effects as the endogenous microRNA molecules [10].

Dharmacon RNAi Technologies (now part of Horizon Discovery) human microRNA miR-142(3p), miR-146a and miR-141 mimics were used to target specific cancer cells through specific transcriptional/posttranscriptional synthetic regulatory circuit [5]. Yan X et al transfected DU145 cells with Thermo Fisher mirVana miRNA mimics (has-miR-34a-5p) to evaluate its effect on CD44 expression [9]. Pandolfini L et al transfected miRIDIAN hsa-let-7e-5p miRNA mimic from Dharmacon (C-300479-05-0002) into A549 cells using lipofectamine 2000 from Thermo Fisher [2]. de Morree A et al transfected purified muscle stem cells using Lipofectamine 2000 with an miR206 mimic and a control mimic to study the effect of miR206 on the Pax3 protein level [1].

QIAGEN miScript Target Protectors are modified single-stranded RNAs that interfere with the interaction of an miRNA with a specific single target gene. Moro A et al, for example, used a miScript Target Protector from QIAGEN directed at the miRNA responsive element (MRE) within the human CTGF gene to disrupt the miRNA–MRE interaction with the CTGF gene to study the role of CTGF in endothelial cell mechanotransduction [11].

MicroRNA inhibitors are able to block the activity of endogenous microRNA molecules, and thus have been used extensively to investigate the function of specific microRNA molecule. de Morree A et al, for example, injected intraperitoneally antisense vivo-morpholino oligonucleotides (AMOs) (from Gene Toos) against miR206 and miR1 into mice and transfected purified mouse muscle stem cells with AMOs using Endoporter from Gene Tools to block their activity in vivo and in vitro [1]. H Kim et al obtained miR-324 inhibitors and their controls from QIAGEN [12].

Most of microRNA research methods such as Northern blot, stem-loop RT-PCR, microarray analysis, and sequencing need purified RNA or enriched small molecule RNA samples.

Important note: TRIzol selectively loses short structured RNAs with low GC content during RNA extraction [13]. It is advisable to avoid using TRIzol for the purification of small RNA in general.

Invitrogen (Ambion) mirVana miRNA Isolation kit can enrich the small RNA molecules and have been used to prepare for qPCR analysis [9, 14] or Northern blot [12]. Lambo S et al extracted total RNA using guanidinium isothiocyanate/phenol from fresh-frozen tumour tissues, ligated barcoded adenylated adaptors and isolated small RNAs (19–35 nt) through gel electrophoresis for miRNA sequencing [15]. Hyun Y et al purified total RNA from plant tissue with MACHEREY-NAGEL NucleoSpin RNA set for NucleoZOL for qPCR analysis of miR156 [3]. Yan X et al used Invitrogen mirVana PARIS RNA Isolation Kit (cat# AM1556) to isolate small RNA-containing total RNA from tumors [9]. Invitrogen TRIzol was commonly used to isolate RNA [16, 17]. Pandolfini L et al extracted total RNA from A549 cells using the miRNeasy Mini kit from QIAGEN and purified RNA using the RNA Clean and Concentrator kit from Zymo Research for small RNA sequencing and PCR [2]. Hsueh MF et al used the saem QIAGEN kit to extract RNA from frozen cartilage tissues to quantify blastema miRNAs (miR-21, miR-31, and miR-181c) through RT-PCR [18].

Yan X et al used Invitrogen TaqMan MicroRNa Reverse Transcription Kit (4366596) to generate cDNA from total RNA obtained from FACS-sorted LAPC4 cells and tumors [9]. Pandolfini L et al used High-Capacity cDNA reverse transcription kit from Applied Biosystems, miScript II RT kit from QIAGEN, and Taqman Advanced miRNA cDNA Synthesis kit from Thermo Fisher Scientific, for the reverse transcription of pri-, pre-, and mature miRNAs, repectively; and used Fast SybrGreen PCR mastermix from Applied Biosystems for the quantitation of pri- and pre-miRNAs, and Taqman advanced miRNA Assays from Thermo Fisher Scientific for that of mature miRNAs [2]. Hsueh MF et al quantified blastema miRNAs (miR-21, miR-31, and miR-181c) from frozen cartilage tissues through RT-PCR with the miScript II RT Kit and the miScript SYBR Green PCR Kit from QIAGEN [18].

Applied Biosystems sequence-specific TaqMan miRNA assay kit was used in real-time PCR to study the expression of miR-34a, let-7b, miR-103 in LAPC4 cells and tumors [9], the expression of two novel/ancient myosins in mammalian skeletal muscles [19], the effect of Hedgehog signals suppression caused by miR-7 on Notch-induced tumourigenesis [20], and the oncogene-dependent apoptosis promoted by a component of the mir-17-92 polycistronic oncomir [16].

One important consideration in PCR analysis of microRNA sample is the internal control. MiRNA quantitative PCR results can be normalized with U6 snRNA / RNU6B [21, 22] or RNY1 [2], or miR-423 and miR-191 [18], or small nucleolar RNA 101 (snoR101) in plant tissue [3]. U6 snRNA (non-coding small nuclear RNA) is a component of the spliceosome where the splicing of pre-mRNA molecule occurs.

Hybridization buffers include Ambion ULTRAhyb-Oligo buffer [23] and Sigma PerfectHyb Plus hybridization buffer [12, 24]. Ambion Nylon Membrane [23] and Amersham Hybond nylon membrane [2, 12] were used.

Northern blot probes can be labeled with gamma-32P-ATP (Amersham Biosciences) by T4 polynucleotide kinase from Takara [12], New England Biolabs [25], or IDT Starfire Labeling Kit [26].

U6 was used as controls for Northern blot analysis [25, 26].

Agilent Human microRNA Microarray (v3) was used to measure the microRNA expression among 14 humans, 11 chimpanzees, and 8 macaques [4].

Invitrogen pDONR207 was used to perform artificial pre-microRNA cloning into miR319a backbone to show that transcription factor KNOTTED1 could be promoted to trafficking through cells by chaperonins [7].

A couple of suppliers dominate the provision of the reagents for miRNA research, as summarized in Table 1. There is a thorough review on RNA extraction reagents. Life Technologies Corporation has several brands prior to its own corporate merger with Thermo Fisher Scientific that provide reagents for miRNA research. The brands include Invitrogen, Applied Biosystems (ABI), Ambion, and Taqman.