Assessment of GAS5 was evaluated by the use of RT2 SYBR Green PCR kit (Qiagen, Maryland, USA). However, the miScript SYBR Green PCR kit (Qiagen, Valenica, CA, USA) was used in the assessment of miR-137 according to the kit instructions with the aid of Rotor gene Q System (Qiagen) on 20 μl reaction mixture.
The RefSeq Accession no. of GAS5 was NR_002578.2 and the catalog number of miR-137 was MS00003486. Assessment of GAS5 was done per the following cycling steps: 95 °C for 10 min, afterward, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. On the other hand, assessment of miR-137 was performed according to the subsequent cycling steps: 95 °C for 30 min, subsequently, 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s.
GAS5 expression values were normalized by the use of GAPDH as an internal control44 (link),45 (link). Though, miR-137 expression values were normalized using SNORD 68 as an internal reference gene. The GAPDH primer sequences were forward, 5′-CCCTTCATTGACCTCAACTA-3′, and reverse 5′-TGGAAGATGGTGATGGGATT-3′. The catalog number of SNORD 68 was MS00033712. Calculation of the fold change (FC) of GAS5 and miR-137 was done with the aid of the equation 2−ΔΔCt46 (link). The FC of control individuals was assumed as 1.
The RefSeq Accession no. of GAS5 was NR_002578.2 and the catalog number of miR-137 was MS00003486. Assessment of GAS5 was done per the following cycling steps: 95 °C for 10 min, afterward, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. On the other hand, assessment of miR-137 was performed according to the subsequent cycling steps: 95 °C for 30 min, subsequently, 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s.
GAS5 expression values were normalized by the use of GAPDH as an internal control44 (link),45 (link)
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