HYBRIDIZATION

The above figure also explains why the four C-H bonds are equivalent to one another. Initially, people thought that if methane had four C-H bonds, that would mean there would be three of one particular type, and one of another type. Hence, this is also something that hybridization explains. Nonetheless, there is even mathematical proof to support this concept! These equations together not only break down what the composition of each hybridized orbital, but also portray


FEATURES
 It is intended for research use only  The total assay time is less than 2.5 hours  The kit measures hsa-miR-23a-3p isolated from human whole blood and cell culture lysates  Assay format is 96 wells  Standard is synthetic miRNA-based  Components of the kit are provided ready to use, concentrated or dried Store the complete kit at 2-8 °C. Under these conditions, all components are stable until the expiration date (see label on the box).
For stability of opened reagents see Chapter 11. MicroRNAs (miRNAs) are small non-coding RNA molecules, approximately 22 nucleotides in length that regulate gene translation through silencing or degradation of target mRNAs. They are involved in multiple biological processes, including differentiation and proliferation, metabolism, hemostasis, apoptosis or inflammation, and in the pathophysiology of many diseases. Numerous studies have suggested circulating miRNAs as promising diagnostic and prognostic biomarkers of many diseases. hsa-miR-23a-3p is located in the miR-23a~27a~24-2 cluster [1]. miR-23a-3p has been reported to be upregulated and have a promoting role in several cancer types via targeting various tumor-suppressor genes [2,3,4,5]. It was also observed that upregulated expression of miR-23a-3p inhibits apoptosis, promotes autophagy and enhances cell colony formation, migration and invasion [6]. Several lines of evidence suggest that dysregulation of miR-23a-3p plays a role in chemoresistance in various types of cancer [7,8]. It was also reported that circulating miR-23a-3p may be involved in postoperative atrial fibrillation development [9]. Decreasedevel of miR-23a-3p was observed in serum of women with polycystic ovary syndrome and could serve as an indicator of this syndrome [10] Moreover, it has been shown that miR-23a-3p plays important roles in myogenesis of skeletal muscle, fiber type determination or exercise adaptation [11]. Overexpression of miR-23-3p could suppress muscle atrophy both in vitro and in vivo [12]. BioVendor hsa-miR-23a-3p miREIA is an enzyme immunoassay for miRNA quantification which involves hybridization of miRNA isolated from a patient sample to complementary biotinylated DNA probe for hsa-miR-23a-3p. The DNA/RNA hybrids are then transferred into microplate wells pre-coated with monoclonal antibody specific to perfectly matched DNA/miRNA hybrids. After washing, the solid phase is incubated with streptavidin-HRP conjugate and after another washing step, the resulting complexes are visualized by chromogenic substrate. The absorbance is proportional to the concentration of hsa-miR-23a-3p. A standard curve is constructed by plotting absorbance values against concentrations of hsa-miR-23a-3p standards. Concentrations of unknown samples are determined using this standard curve.

Sample Type
hsa-miR-23a-3p miREIA is intended for miRNA isolated from human whole blood and cell culture lysates.
Ask for information at info@biovendor.com if assaying miRNA isolated from another material.

Processing of whole blood
Conditions during sample collection may affect the detection of microRNAs. Therefore, it is highly recommended to follow standardized procedure for blood collection:  To minimize patient variables, it is recommended to ensure overnight fasting prior to blood collection. Circadian rhythm, activity and diet are known to influence the microRNA levels  Standardized needles and blood collection tubes are needed  Gloves must be worn all the time when handling specimens  PAXgene Blood RNA Tubes are recommended for whole blood collection and storage. Follow the instructions for blood collection and handling provided by the manufacturer: https://www.preanalytix.com/  Immediately after blood collection, gently invert the PAXgene RNA tubes 10 times, then let the tubes stand in upright position for at least

Spike-In Control
It is recommended to normalize measured concentrations of hsa-miR-23a-3p by exogenous control. The concentration of miRNA measured by miREIA can be affected by efficiency of RNA isolation. For monitoring the efficiency of isolation, it is recommended to add a defined amount of synthetic nonhuman RNA Spike-In Control to the samples after addition of lysis buffer e.g. cel-miR-39-3p. Each laboratory should establish its own dilution factor of normal and pathological samples for hsa-miR-23a-3p levels with the assay.
Note: It is recommended to use a precise pipette and a careful technique to perform the dilution in order to get precise results.

PREPARATION OF HYBRIDS
The procedure described below must be performed for each point of the standard curve, sample and blank separately (see the Hybridization Procedure Summary below).

HYBRIDIZATION PROCEDURE
Insert nuclease-free tubes with the hybrids prepared in the previous step into a cycler and run the hybridization program. It is also possible to use a thermoblock instead of the cycler.  Figure 1). Covering the plate with e.g. microplate cover or sealing film is recommended. 3. Incubate the plate at 37 °C for 1 hour, without shaking. 4. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel. 5. Add 100 l of Streptavidin-HRP Conjugate into each well. Covering the plate with e.g microplate cover or sealing film is recommended. 6. Incubate the plate at 37 °C for 30 minutes, without shaking. 7. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel. 8. Add 100 l of Substrate Solution into each well. Avoid exposing the microtiter plate to direct sunlight. Covering the plate with e.g. aluminium foil is recommended. 9. Incubate the plate for 15 minutes at room temperature. The incubation time may be extended [up to 30 minutes] if the reaction temperature is below 20 °C. Do not shake the plate during the incubation. 10. Stop the colour development by adding 100 l of Stop Solution. 11. Determine the absorbance of each well using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550-650 nm). Subtract readings at 630 nm (550-650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 10.
Note 1: If some samples and standard/s have absorbances above the upper limit of your microplate reader, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine hsa-miR-23a-3p concentration of off-scale standards and samples. The readings at 405 nm should not replace the readings for samples that were "in range" at 450 nm.
Note 2: Manual washing 5-times: Aspirate wells and pipet 0.35 ml Wash Solution into each well. Aspirate wells and repeat four times. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

Sensitivity
Limit of detection (LOD) (defined as concentration of analyte giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank: Ablank + 3xSDblank is calculated from the real hsa-miR-23a-3p values in wells and is 0.13 amol/l.

Limit of Assay
Samples with absorbances exceeding the absorbance of the highest standard should be measured again with higher dilution. The final concentration of samples calculated from the standard curve must be multiplied by the respective dilution factor.

Specificity
The DNA Probe for hsa-miR-23a-3p is complementary to the sequence of hsa-miR-23a-3p. Crossreactivity with the miRNA family members exhibiting high sequence identity cannot be excluded

Reference range
It is recommended that each laboratory include its own panel of control samples in the assay. Each laboratory should establish its own normal and pathological reference ranges for hsa-miR-23a-3p levels with the assay. The synthetic hsa-miR-23a-3p is used as the standard for hsa-miR-23a-3p quantification.

Weak signal in all wells
Possible explanations:  Omission of a reagent or a step  Improper preparation or storage of a reagent  Assay performed before reagents were allowed to come to room temperature  Improper wavelength when reading absorbance