circRNA Supporting Data

A) Tested three circRNAs for over-expression in HEK293T cells

circRNA 1, 2 and 3 have been documented to be expressed in many tissues; notably circRNA 1 is expressed in kidney tissues (and thus in HEK293T cells) whereas circRNA 2 and 3 are not.

	circRNA1	circRNA2	circRNA3
Expression Tissues	Kidney, Liver, Brain, Small Intestine, Stomach, Lung, Spleen, Thymus, Placenta, Colon, Bone Marrow	Brain, Liver, Placenta, Colon, Heart, Spinal Cord, Uterus, Prostate, Lymph node, Tonsil, Blood	Liver, Prostate, Colon, Brain, Bone Marrow, Testis, Breast, Lung
Associated Diseases	N/A	Breast cancer, clear cell renal cell carcinoma, epithelial ovarian cancer, glioblastoma, hepatocellular carcinoma	Bladder cancer, hepatocellular carcinoma, renal cell carcinoma, hepatocellular carcinoma
circRNA Type	5’ UTR	Exonic	Exonic

B) Cloned circRNAs into abm’s circRNA Expression Vector

The circRNA Expression Vector utilizes an optimized complementary sequence-mediated circRNA circularization strategy – these sequences work to bring the exon flanking portion of introns in close proximity thus promoting highly efficient back-splicing into circRNA in vivo.

C) Transfected circRNA Expression Vectors into HEK293T cells

The circRNA Expression Vectors contain an eGFP cassette allowing for convenient assessment of transfection efficiency.

abm products used: DNAfectin™ Plus Transfection Reagent (Cat. No. G2500)

D) Extracted total RNA from transfected cells

Total RNA was analyzed for integrity and purity by running samples on an agarose gel.

abm products used:
Column-Pure RNA Miniprep Kit (Cat. No. D518)
SafeView™ Classic Nucleic Acid Stain (Cat. No. G108)

E) Total RNA was subjected to RT-qPCR using divergent primers

The standard method to detect circRNA expression is by RT-qPCR using divergent primers. These primers are designed in opposing directionality, and thus will only create a qPCR amplicon if correct circularization has occurred.

abm products used:
OneScript® Hot cDNA Synthesis Kit (Cat. No. G594)
BlasTaq™ 2X qPCR MasterMix (Cat. No. G891)

circRNA1 was successfully over-expressed in HEK293T cells. Cells transfected with Empty Vector Control (red) or circRNA1 Expression Vector (green) were compared. Earlier CT values and amplification curves of circRNA1 indicated successful over-expression of circRNA1 in HEK293T cells. Melt curve analysis indicated the amplicon detected was the same circRNA species in both transfected cell groups, confirming endogenous expression of circRNA1.

circRNA2 was successfully ectopically expressed in HEK293T cells. Cells transfected with Empty Vector Control (red) or circRNA2 Expression Vector (green) were compared. Amplification curve of circRNA2 only (green) indicated correct circRNA expression and no endogenous expression of this species in HEK293T cells.

circRNA3 was successfully ectopically expressed in HEK293T cells. Cells transfected with Empty Vector Control (red) or circRNA3 Expression Vector (green) were compared. Amplification curve of circRNA3 only (green) indicated correct circRNA expression and no endogenous expression of this species in HEK293T cells.