3.1 Expression levels of the candidate reference miRNA
The cycle threshold (Ct) value for each reference miRNA is presented in Table S1. There were significant differences across the 16 reference miRNAs in the ten body fluids. The mean Ct value of the candidate genes ranged from 22 to 40 cycles, with miR-148a having the highest transcript levels (22.73 ± 0.02) in ordinary milk and miR-16 with the lowest (39.49 ± 0.14) in urine. Based on the geNorm calculations of the preliminary Ct values and stability sequencing (Table S2), ten miRNAs were selected for subsequent stability analysis including, miR-92a, miR-93, let-7a, miR-451, miR-106a, U6, miR-17-5P, miR-26a, miR-25, let-7b-5p.
3.2 GeNorm analysis of candidate reference miRNAs in body fluids
The geNorm analysis revealed that all candidate genes had an M value below 1.5, which is the maximum value for acceptable gene expression stability [26]. The gene with the lowest M value has the most stable expression. However, the optimal candidate miRNA stability expressed in each body fluid differed (Table S2). The most stable miRNA were miR-25 and let-7b-5p in URBP, let-7b-5p and miR-93 in UVBP, miR-26a and miR-335 in PBS, miR-17-5P and miR-148a in ordinary milk, miR-451 and miR-16 in colostrum, miR-103 and miR-451 in semen, miR-191 and miR-92a in urine, let-7b-5p and miR-93 in bile, miR-191 and miR-16 in bladder fluid, and miR-17-5p and miR-93 in gastric juice. Further analyses of the M value ranking in all body fluids in vivo, in vitro, and the blood revealed the first five ranking of stability in all body fluids as miR-451 > miR-92a (let-7b-5p) > miR-106a (U6). The average stability rank in the blood, in vitro and in vivo was let-7b-5p > miR-25 > U6, miR-451 > miR-92a > miR-106a, and U6 > miR-92a > let-7a, respectively.
3.3 Analysis of candidate reference miRNAs in blood
To explore the stability of miRNA expression, the stability of miRNA in URBP, UVBP, and PBS were analyzed (Table 1). A comprehensive ranking of the miRNA expression stability revealed that let-7b-5p expressed in URBP was the most stable, ranking 1, 2, and 1 with GeNorm, NormFinder, and BestKeeper, respectively. Moreover, miR-25 expression was the most stable in UVBP and PBS. The U6 expression was also stable in PBS.
Table 1
Ranking and best combination of candidate reference genes in blood
URBP
|
Genormer (M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
UVBP
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
PBS
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
miR-92a
|
0.12(10)
|
0.11(10)
|
0.27(8)
|
miR-17-5P
|
0.10(10)
|
0.09(10)
|
0.03(2)
|
miR-451
|
0.11(10)
|
0.11(10)
|
0.33(10)
|
miR-93
|
0.11(9)
|
0.10(9)
|
0.32(9)
|
miR-92a
|
0.09(9)
|
0.08(9)
|
0.31(8)
|
miR-106a
|
0.09(9)
|
0.10(9)
|
0.29(9)
|
let-7a
|
0.10(8)
|
0.09(8)
|
0.34(10)
|
miR-26a
|
0.08(8)
|
0.08(8)
|
0.31(7)
|
miR-93
|
0.08(8)
|
0.08(8)
|
0.24(8)
|
miR-451
|
0.08(7)
|
0.07(7)
|
0.23(7)
|
miR-106a
|
0.07(7)
|
0.04(5)
|
0.19(5)
|
miR-92a
|
0.06(7)
|
0.04(6)
|
0.12(6)
|
miR-106a
|
0.06(6)
|
0.05(6)
|
0.12(5)
|
miR-451
|
0.07(6)
|
0.04(6)
|
0.31(9)
|
let-7b-5p
|
0.06(6)
|
0.05(7)
|
0.13(7)
|
U6
|
0.06(5)
|
0.04(5)
|
0.11(4)
|
miR-93
|
0.06(5)
|
0.02(1)*
|
0.20(6)
|
miR-26a
|
0.04(5)
|
0.01(3)
|
0.03(2)
|
miR-17-5P
|
0.04(4)
|
0.01(1)*
|
0.19(6)
|
let-7b-5p
|
0.06(4)
|
0.02(2)
|
0.16(4)
|
let-7a
|
0.03(4)
|
0.01(2)
|
0.10(3)
|
miR-26a
|
0.03(3)
|
0.04(4)
|
0.10(3)
|
U6
|
0.04(3)
|
0.05(7)
|
0.15(3)
|
U6*
|
0.03(3)
|
0.01(1)*
|
0.02(1)*
|
miR-25
|
0.02(1)*
|
0.02(3)
|
0.09(2)
|
miR-25*
|
0.03(1)*
|
0.03(3)
|
0.01(1)*
|
miR-25*
|
0.01(1)*
|
0.04(5)
|
0.11(4)
|
let-7b-5p*
|
0.02(1)*
|
0.01(2)
|
0.08(1)*
|
let-7a
|
0.03(1)*
|
0.03(4)
|
0.38(10)
|
miR-17-5P
|
0.01(1)*
|
0.04(4)
|
0.12(5)
|
Note: Umbilical arterial blood plasma (URBP),Umbilical venous blood plasma(UVBP), Peripheral blood serum(PBS), * represents the ranking and the best combination of candidate reference genes. The numbers in brackets represent the stability sort.
|
3.4 Analysis of the candidate reference miRNAs in body fluids in vitro
Except for blood, commonly used to detect disease, body fluids in vitro are more readily available than in vivo. Here, the stability of miRNA expression in body fluids in vitro (ordinary milk, colostrum, semen, and urine) was analyzed. The findings are presented in Table 2. A comprehensive ranking presented miR-106a, let-7b-5p, miR-25, and miR-92a as the most stably expressed in ordinary milk (ranks 1, 1 and 3), colostrum (ranks 1, 1 and 1), semen (ranks 5, 1 and 3), and urine (ranks 1, 2 and 1) using GeNorm, NormFinder, and BestKeeper, respectively.
Table 2
Ranking and best combination of candidate reference genes of body fluids in vitro
Ordinary milk
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
Colostrum
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
Semen
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
Urine
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
let-7a
|
0.06(10)
|
0.07(10)
|
0.36(9)
|
let-7a
|
0.09(10)
|
0.08(9)
|
0.37(10)
|
let-7b-5p
|
0.12(10)
|
0.12(10)
|
0.27(8)
|
U6
|
0.11(10)
|
0.15(10)
|
0.38(9)
|
miR-25
|
0.05(9)
|
0.06(9)
|
0.25(8)
|
miR-25
|
0.08(9)
|
0.08(10)
|
0.22(8)
|
let-7a
|
0.10(9)
|
0.08(9)
|
0.25(7)
|
miR-25
|
0.08(9)
|
0.10(9)
|
0.29(7)
|
U6
|
0.05(8)
|
0.05(8)
|
0.2(7)
|
miR-93
|
0.07(8)
|
0.08(8)
|
0.22(9)
|
miR-93
|
0.09(8)
|
0.08(8)
|
0.24(5)
|
let-7a
|
0.07(8)
|
0.08(8)
|
0.30(8)
|
miR-26a
|
0.04(7)
|
0.03(6)
|
0.07(1)*
|
miR-26a
|
0.06(7)
|
0.06(7)
|
0.13(7)
|
miR-92a
|
0.08(7)
|
0.04(4)
|
0.10(2)
|
miR-106a
|
0.06(7)
|
0.05(7)
|
0.10(4)
|
miR-93
|
0.04(6)
|
0.03(7)
|
0.12(6)
|
miR-17-5P
|
0.05(6)
|
0.03(5)
|
0.08(4)
|
miR-451
|
0.08(6)
|
0.03(3)
|
0.08(1)*
|
miR-26a
|
0.05(6)
|
0.03(6)
|
0.05(2)
|
let-7b-5p
|
0.03(5)
|
0.02(5)
|
0.10(4)
|
miR-106a
|
0.04(5)
|
0.01(2)
|
0.07(3)
|
miR-25*
|
0.07(5)
|
0.01(1)*
|
0.11(3)
|
let-7b-5p
|
0.04(5)
|
0.02(5)
|
0.11(6)
|
miR-17-5P
|
0.02(4)
|
0.01(3)
|
0.37(10)
|
miR-92a
|
0.04(4)
|
0.05(6)
|
0.11(6)
|
miR-26a
|
0.06(4)
|
0.01(1)*
|
0.14(4)
|
miR-93
|
0.04(4)
|
0.02(4)
|
0.11(5)
|
miR-451
|
0.02(3)
|
0.02(4)
|
0.11(5)
|
U6
|
0.03(3)
|
0.03(4)
|
0.09(5)
|
miR-17-5P
|
0.03(3)
|
0.08(7)
|
0.32(10)
|
miR-451
|
0.03(3)
|
0.01(1)*
|
0.09(3)
|
miR-106a*
|
0.01(1)*
|
0.01(1)*
|
0.08(3)
|
miR-451
|
0.02(2)
|
0.02(3)
|
0.02(2)
|
miR-106a
|
0.02(1)*
|
0.05(5)
|
0.24(6)
|
miR-92a*
|
0.02(1)*
|
0.01(2)
|
0.01(1)*
|
miR-92a
|
0.01(1)*
|
0.01(2)
|
0.07(2)
|
let-7b-5p*
|
0.02(1)*
|
0.01(1)*
|
0.02(1)*
|
U6
|
0.02(1)*
|
0.06(6)
|
0.32(9)
|
miR-17-5P
|
0.02(1)*
|
0.01(3)
|
0.38(10)
|
Note: * represents the ranking and the best combination of candidate reference genes.
|
3.5 Analysis of the candidate reference miRNAs in body fluids in vivo
The stability of miRNA expressed in body fluids in vitro, including in bile, bladder fluid, and gastric juice, is presented in Table 3. A comprehensive ranking of the miRNA expression stability revealed that miR-93 (let-7b-5p), miR-92a, and miR-93 were most stably expressed in bile (ranks 1, 2, and 1), bladder fluid (ranks 5, 2, and 2), and gastric juice (ranks 1, 2 and 2) based on the GeNorm, NormFinder and BestKeeper analyses, respectively.
Table 3
Ranking and best combination of candidate reference genes of body fluids in vivo
Bile
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
Bladder fluid
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
Gastric juice
|
Genormer(M value)
|
Normfinder Stability value
|
Bestkeeper CV [% CP]
|
miR-451
|
0.12(10)
|
0.11(10)
|
0.40(10)
|
miR-93
|
0.11(10)
|
0.11(10)
|
0.28(10)
|
miR-26a
|
0.07(10)
|
0.09(10)
|
0.27(10)
|
miR-25
|
0.11(9)
|
0.10(9)
|
0.28(9)
|
miR-17-5P
|
0.10(9)
|
0.10(9)
|
0.13(3)
|
let-7b-5p
|
0.05(9)
|
0.08(9)
|
0.20(9)
|
miR-17-5P
|
0.09(8)
|
0.07(8)
|
0.10(3)
|
let-7b-5p
|
0.08(8)
|
0.08(7)
|
0.17(6)
|
let-7a
|
0.04(8)
|
0.03(7)
|
0.10(6)
|
miR-92a
|
0.09(7)
|
0.03(3)
|
0.15(6)
|
miR-25
|
0.08(7)
|
0.08(8)
|
0.25(9)
|
miR-25
|
0.04(7)
|
0.03(8)
|
0.13(8)
|
miR-26a
|
0.08(6)
|
0.06(4)
|
0.15(7)
|
U6
|
0.07(6)
|
0.03(3)
|
0.04(1)*
|
U6
|
0.03(6)
|
0.02(6)
|
0.07(4)
|
miR-106a
|
0.07(5)
|
0.07(7)
|
0.15(5)
|
miR-92a*
|
0.06(5)
|
0.02(2)
|
0.06(2)
|
miR-92a
|
0.03(5)
|
0.02(5)
|
0.08(5)
|
U6
|
0.05(4)
|
0.06(6)
|
0.15(4)
|
let-7a
|
0.04(4)
|
0.01(1)*
|
0.19(8)
|
miR-451
|
0.02(4)
|
0.01(1)*
|
0.02(1)*
|
let-7a
|
0.04(3)
|
0.06(5)
|
0.22(8)
|
miR-451
|
0.01(3)
|
0.03(4)
|
0.15(4)
|
miR-106a
|
0.02(3)
|
0.01(3)
|
0.04(3)
|
let-7b-5p*
|
0.04(1)*
|
0.03(2)
|
0.04(1)*
|
miR-106a
|
0.01(1)*
|
0.04(5)
|
0.17(5)
|
miR-93*
|
0.02(1)*
|
0.01(2)
|
0.04(2)
|
miR-93*
|
0.04(1)*
|
0.02(1)*
|
0.05(2)
|
miR-26a
|
0.01(1)*
|
0.04(6)
|
0.18(7)
|
miR-17-5P
|
0.02(1)*
|
0.01(4)
|
0.12(7)
|
Note: * represents the ranking first and the best combination of candidate reference miRNAs.
|
3.6 Comprehensive ranking of the reference miRNAs
Given that each body fluid has its distinct characteristics, it is necessary to identify the optimal reference miRNAs in each fluid for a better practical application. The comprehensive comparative analysis revealed that miR-25, let-7b-5p, miR-25, and U6 were the optimal reference miRNA in blood, URBP, UVBP, and PBS, respectively (Table 4). The optimal reference miRNA in body fluids obtained in vitro was miR-451 and miR-92a, with miR-92a, let-7b-5p, miR-25, and miR-92a as the optimal reference miRNA in ordinary milk, colostrum, semen, and urine, respectively. In addition, the optimal reference miRNA in body fluids in vivo was miR-93, with let-7b-5p (miR-93), miR-92a, and miR-93 as the optimal reference miRNA in bile, bladder fluid, and gastric juice, respectively. Overall, miR-25, miR-92, and miR-93 were the most common in blood, in vivo, and in vitro, respectively.
Table 4
The Optimal reference gene in each body fluids calculated by geNorm, NormFinder and Bestkeeper algorithms
Type of fluid
|
URBP
|
UVBP
|
PBS
|
Blood
|
Ordinary milk
|
Colostrum
|
Semen
|
Urine
|
Body fluids in vitro
|
Bile
|
Bladder fluid
|
Gastric juice
|
Body fluids in vivo
|
Optimal reference gene
|
let-7b-5p
|
miR-25
|
U6
|
miR-25
|
miR-92a
|
let-7b-5p
|
miR-25
|
miR-92a
|
miR-451 miR-92a
|
let-7b-5p
miR-93
|
miR-92a
|
miR-93
|
miR-93
|
Note: Umbilical arterial blood plasma (URBP), Umbilical venous blood plasma (UVBP), Peripheral blood serum (PBS).
|