Exploration of Rhythmic Patterns of Gene Expression to Estimate the Time of Day a Bloodstain Was Created

Figures & data

Table

Dénaturation		Amplification – 40 Cycles			Disassociation
50°C 2 min	95°C 2 min	95°C 15 sec	58°C 15 sec	72°C 1 min	95°C 15 sec	60°C 1 min

Table 1 qPCR Primers to Quantify Expression of Genes During the Circadian Rhythm

mRNA	Primer Sequence	Tm	Product Length
Per1 F Per1 R	5′ GAATACTACCAGCAGTGGAG 3′ 5′ GTATCCTGGTTCTGAAGTGTG 3′	55°C 56°C	119bp
Per2 F Per2 R	5′ GAAATCCGCTACCACCCCT 3′ 5′ GCTTCATAACCAGAGTGCAC 3′	57°C 56°C	121bp
Per3 F Per3 R	5′ GAGACGCAATAAACCAAGCA 3′ 5′ CTGAGGTGCTCCATTCTGAC 3′	57°C 58°C	115bp
S100A12 F S100A12 R	5′ TCCAAGGCCTGGATGCTAATC 3′ 5′ TGTGGTAATGGGCAGCCTTC 3′	60°C 60°C	93bp
BMAL1 F BMAL1 R	5′ CCCATTGAACATCACGAGTAC 3′ 5′ GAGCCTGGCCTGATAGTAG 3′	62°C 60°C	116bp
CLOCK F CLOCK R	5′ CCAACCCCTTCTGCCTCTT 3′ 5′ GTAAATGCTGCCTGGGTGG 3′	59°C 59°C	121bp
Cry1 F Cry1 R	5′ TTGGAAAGGAACGAGACGC 3′ 5′ CGGTTGTCCACCATTGAGTT 3′	58°C 58°C	125bp
Cry2 F Cry2 R	5′ GTGTGGAAGTAGTGACGGA 5′ GCTGATGATGGCCTGAAAG 3′	56°C 56°C	113bp
CyE F CyE R	5′ GTTGCACCAGTTTGCGTATG 3′ 5′ GCCAGGACACAATAGTCAG 3′	60°C 54°C	122bp
MKNK2 F MKNK2 R	5′ CATAACAAAGGCATCGCCC 3′ 5′ GTTGAGTTTGATGCCGCTG 3′	58°C 58°C	120bp
THRAI F THRAI R	5′ GTGTGGGGACAAGGCAAC 3′ 5′ CTGTCATATTTGCAGGAATAG 3′	58°C 56°C	120bp
TPOHI F FTPOHI R	5′ GCTGTTCAAAAACTGGCAAC 3′ 5′ GAAGAAAGTAAGCCAGCACC 3′	58°C 60°C	101bp

Table 2 Variation in Transcript Abundance Levels in Bloodstains Collected from a Single Individual at the Same Time on Different Days*

mRNA	Primer Sequence	Tm	Product Length
Per1 F Per1 R	5′ GAATACTACCAGCAGTGGAG 3′ 5′ GTATCCTGGTTCTGAAGTGTG 3′	55°C 56°C	119bp
Per2 F Per2 R	5′ GAAATCCGCTACCACCCCT 3′ 5′ GCTTCATAACCAGAGTGCAC 3′	57°C 56°C	121bp
Per3 F Per3 R	5′ GAGACGCAATAAACCAAGCA 3′ 5′ CTGAGGTGCTCCATTCTGAC 3′	57°C 58°C	115bp
S100A12 F S100A12 R	5′ TCCAAGGCCTGGATGCTAATC 3′ 5′ TGTGGTAATGGGCAGCCTTC 3′	60°C 60°C	93bp
BMAL1 F BMAL1 R	5′ CCCATTGAACATCACGAGTAC 3′ 5′ GAGCCTGGCCTGATAGTAG 3′	62°C 60°C	116bp
CLOCK F CLOCK R	5′ CCAACCCCTTCTGCCTCTT 3′ 5′ GTAAATGCTGCCTGGGTGG 3′	59°C 59°C	121bp
Cry1 F Cry1 R	5′ TTGGAAAGGAACGAGACGC 3′ 5′ CGGTTGTCCACCATTGAGTT 3′	58°C 58°C	125bp
Cry2 F Cry2 R	5′ GTGTGGAAGTAGTGACGGA 5′ GCTGATGATGGCCTGAAAG 3′	56°C 56°C	113bp
CyE F CyE R	5′ GTTGCACCAGTTTGCGTATG 3′ 5′ GCCAGGACACAATAGTCAG 3′	60°C 54°C	122bp
MKNK2 F MKNK2 R	5′ CATAACAAAGGCATCGCCC 3′ 5′ GTTGAGTTTGATGCCGCTG 3′	58°C 58°C	120bp
THRAI F THRAI R	5′ GTGTGGGGACAAGGCAAC 3′ 5′ CTGTCATATTTGCAGGAATAG 3′	58°C 56°C	120bp
TPOHI F FTPOHI R	5′ GCTGTTCAAAAACTGGCAAC 3′ 5′ GAAGAAAGTAAGCCAGCACC 3′	58°C 60°C	101bp

Figure 1 Mean abundance values (expressed as (ΔCq)) for circadian panel transcript markers (labeled atop each row of datapoints in the figure) for each age group of males and females normalized against S100A12 transcript.

Figure 2 Mean abundance values (expressed as (ΔCq)) for circadian panel transcript markers (labeled atop each row of datapoints in the figure) for each age group of males and females normalized against CLOCK transcript.

Table 3 Relative Expression of Circadian Rhythm Genes in Blood Stains from Males versus Females with Δcq Values Produced Using S100A12 or CLOCK*

Markers Evaluated for Female vs Male Expression	p-value; Data Normalized to S100A12	p-value; Data Normalized to CLOCK
PER1	0.9910	<0.0001
PER2	>0.9999	0.006
PER3	>0.9999	<0.0001
BMAL1	>0.9999	0.9998
CLOCK	0.9519	NA
CRY1	0.9922	>0.9999
CRY2	>0.9999	0.0363
CyE	>0.9999	0.9998
MKNK2	0.9992	0.0112
THRA1	>0.9999	0.0539
TPOH	>0.9999	0.8912

Figure 3 24-hour expression of PER3 (normalized to CLOCK) in blood stains collected from females every 4 hours for 24 hours. Standard error bars indicate the variance in normalized ΔCq values at sample collection times during a 24-hour period.

Figure 4 24-hour expression of PER3 (normalized to CLOCK) in blood stains collected from males every 4 hours for 24 hours. Standard error bars indicate the variance in normalized ΔCq values at sample collection times during a 24-hour period. The higher variance of PER3 ΔCq values in males obscures any abundance pattern.