TITLE: Association between circulating microRNA-122, microRNA-126-3p and microRNA-146a and inflammatory markers in patients with pre-diabetes and type 2 diabetes mellitus
AUTHORS: Hassan Mozaffari-Khosravi, Fahime Zeinali, Seyed Mohsen Aghaei Zarch, Alireza Jahan-mihan, Shima Abdollahi, Seyed Mehdi Kalantar, Mohammad Yahya Vahidi Mehrjardi, Hossein Fallahzadeh, Mahdieh Hosseinzadeh-Shamsi-Anar, Masoud Rahmanian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Ninety individuals were included in this case-control study:30 type 2 diabetes mellitus, 30 pre-diabetes and 30 healthy individuals (males and females, age: 25-65, body mass index: 25-35). MicroRNAs expression was determined by real-time polymerase chain reaction. Furthermore, plasma concentrations of tumor necrosis factor alpha (TNF-α) and interleukin 6 (Il-6) and fasting insulin was measured by enzyme-linked immunosorbent assay. Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated as an indicator of insulin resistance.</div></div>

[STEPS]
?. [Real time PCR]
Real time PCR
?. [IL6 measurement]
IL6 measurement by ELISA kit https://www.diaclone.com/documents/protocole/950.030_Human_IL-6_ELISA_kit_insert_v11.pdf
?. [RNA extraction]
RNA extraction http://tribioscience.com/files/315-150.pdf
?. [RNA extraction]
Prepare 750 ul RiboExTM LS in a 1.5 ml microcentrifuge tube (not provided).
?. [RNA extraction]
Add 250 ul blood sample to the 1.5 ml microcentrifuge tube and vortex vigorously. If sample volume is 100 ul, sample should be adjusted to 250 ul with PBS or RNase-free water. Be sure to confirm the applicable minimum volume, which is 100 ul.
?. [RNA extraction]
Incubate 2 min at room temperature. This step allows leukocytes to completely be collapsed.
?. [RNA extraction]
Add 0.2 ml of chloroform. Shake vigorously for 15 seconds and let it stand for 2 minutes at room temperature. Alternatively, 0.1 ml of BCP (1-bromo-3-chloropropane) can be used in place of chloroform.
?. [RNA extraction]
Centrifuge at 12,000 x g for 15 minutes at 4°C. The mixture will be separated into three phases; a lower layer, an interphase, and a colorless upper aqueous layer. The upper aqueous volume is about 450 ul. Centrifugation at temperatures >8°C may cause some DNA to partition in the aqueous phase.
?. [TNF alpha measurement]
Read the absorbance value of each well (immediately after step 5.11.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 620 nm as the reference wave length (610 nm to 650 nm is acceptable).
?. [TNF alpha measurement]
Addition: Add 100μl of H2SO4:Stop Reagent into all wells
?. [TNF alpha measurement]
Incubation: Incubate in the dark for 12-15 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
?. [TNF alpha measurement]
Addition: Add 100μl of ready-to-use TMB Substrate Solution into all wells
?. [TNF alpha measurement]
Wash: Repeat wash step 5.5.
?. [TNF alpha measurement]
Incubation: Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 30 min
?. [TNF alpha measurement]
Addition: Add 100μl of Streptavidin-HRP solution into all wells
?. [TNF alpha measurement]
Wash: Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.3 ml of 1x wash buffer into each well c) Aspirate the contents of each well d) Repeat step b and c another two times
?. [TNF alpha measurement]
Incubation: Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 3 hours
?. [TNF alpha measurement]
Addition: Add 50l of diluted biotinylated anti-TNF to all wells
?. [TNF alpha measurement]
Addition: Add 100l of each Sample, Standard, Control and zero (appropriate standard diluent) in duplicate to appropriate number of wells
?. [TNF alpha measurement]
Addition: Prepare Standard curve
?. [TNF alpha measurement]
TNF alpha measurement by ELISA kithttps://www.diaclone.com/documents/protocole/950.090_Human_TNFa_ELISA_kit_version_9.pdf
?. [IL6 measurement]
Addition: Prepare Standard curve
?. [IL6 measurement]
Addition :Add 100µl of each Sample, Control and zero (Standard diluent) in duplicate to appropriate number of wells
?. [IL6 measurement]
Addition: Add 50µl of diluted biotinylated anti-IL-6 to all wells
?. [IL6 measurement]
Incubation: Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour
?. [IL6 measurement]
Wash: Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.3 ml of 1x washing solution into each well c) Aspirate the contents of each well d) Repeat step b and c another two times
?. [IL6 measurement]
Addition: Add 100μl of Streptavidin-HRP solution into all wells
?. [IL6 measurement]
Incubation: Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 30 min
?. [IL6 measurement]
Wash: Repeat wash step 4.5.
?. [IL6 measurement]
Addition: Add 100μl of ready-to-use TMB Substrate Solution into all wells
?. [IL6 measurement]
Incubation: Incubate in the dark for 12-15 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
?. [IL6 measurement]
Addition :Add 100μl of H2SO4:Stop Reagent into all wells Read the absorbance value of each well (immediately after step 4.11.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 620 nm as the reference wave length (610 nm to 650 nm is acceptable).
?. [Fasting insulin]
Determining serum levels of fasting insulin by ELISA kit
?. [Fasting insulin]
Addition: Prepare Standard curve
?. [Fasting insulin]
Addition: Add 50l of each Sample, Standard, Control and zero (appropriate standard diluent) in duplicate to appropriate number of wells
?. [Fasting insulin]
Addition: Add 100l of diluted conjugate Insulin-R to all wells
?. [Fasting insulin]
Shake the plate for 20-30 seconds in room temperature
?. [Fasting insulin]
Incubation: Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour
?. [Fasting insulin]
Wash: Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.35 ml of 1x wash buffer into each well c) Aspirate the contents of each well d) Repeat step b and c another two times
?. [Fasting insulin]
Addition: Add 100μl of substrate solution into all wells
?. [Fasting insulin]
Incubation: Incubate in the dark for 15 minutes at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
?. [Fasting insulin]
Addition: Add 100μl of Stop Reagent into all wells. Put the plate on the table without any shake for 15-20 seconds.
?. [Fasting insulin]
Read the absorbance value of each well (immediately after step 6.9.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 620 nm as the reference wave length.
?. [cDNA synthesis]
cDNA synthesis
?. [cDNA synthesis]
Prepare the following reaction mixture in a microcentrifuge tube to the total volume of 12µl:   AB1  Total RNA    1-5 µg  2  BON RT adaptor    1 µl  3  RNase free water    Up to 12 µl
AB1  Total RNA    1-5 µg  2  BON RT adaptor    1 µl  3  RNase free water    Up to 12 µl
?. [cDNA synthesis]
Incubation in thermocycler at 75 °C for 5 minute.
?. [cDNA synthesis]
Place on ice immediately and add following component in bellow table.  AB1  RT enzyme    1 µl  2  dNTP    2 µl  3  5×RT Buffer    4 µl  4  RNase free water    Up to 20 µl
AB1  RT enzyme    1 µl  2  dNTP    2 µl  3  5×RT Buffer    4 µl  4  RNase free water    Up to 20 µl
?. [cDNA synthesis]
Incubation the mixture in thermocycler according to following temperature and duration and cycle:  ABC1  25 °C    10 min    1 cycle  2  42 °C    60 min    1 cycle  3  70 °C    10 min    1 cycle
ABC1  25 °C    10 min    1 cycle  2  42 °C    60 min    1 cycle  3  70 °C    10 min    1 cycle
?. [Real time PCR]
After cDNA synthesis, add the following components:  AB1  cDNA    1 µl  2miRNA specific forward primer  0.5 µl  3universal reverse primer  0.5 µl  4  2×miRNA QPCR master mix    6.5 µl  5Nuclease free PCR grade H2O  Up to 13 µl
AB1  cDNA    1 µl  2miRNA specific forward primer  0.5 µl  3universal reverse primer  0.5 µl  4  2×miRNA QPCR master mix    6.5 µl  5Nuclease free PCR grade H2O  Up to 13 µl
?. [Real time PCR]
Incubation the mixture in thermocycler according to following temperature and duration and cycle:  ABC1  95 °C    2 min    1 cycle  2  95 °C    5 sec       3  60 °C    30 sec    40 cycle
ABC1  95 °C    2 min    1 cycle  2  95 °C    5 sec       3  60 °C    30 sec    40 cycle