twenty - one premature infants born between 30 and 34 wk ga with a birth weight > 1500 g were recruited for this study . exclusion criteria included major chromosomal anomalies , intraventricular hemorrhage ( > grade ii ) , necrotizing enterocolitis , excessive drug / alcohol use , data missing regarding the date to full oral feeds , and/or the need for prolonged respiratory support ( i.e. , high - flow nasal cannula , continuous positive airway pressure , or ventilator support ) that would have delayed oral feeding trials . saliva samples were taken at the closest approximate time ( within the 3234-wk period ) that an infant commenced oral feeding attempts . there were a few infants ( see those indicated by an   in table 1 ) that attained full oral feeds before the acquisition of the salivary sample . two of these infants were capable of full oral feeds from birth , and the other took 5 d to attain full oral feeds , only briefly requiring nasogastric support before the attainment of a sample . all infants in the tufts medical center nicu are subjectively assessed for oral feeding readiness based on the cue - based feeding algorithm described by ludwig and waitzman ( 2007 ) . salivary samples were collected and processed with previously described techniques in order to simulate routine bedside care of the neonates ( dietz et al . the neonate 's oropharynx was gently suctioned ( < 1 min ) and saliva was immediately stabilized in 500 l of rnaprotect saliva ( qiagen ) . this stabilizing agent halts gene expression changes , inhibits microbial overgrowth , and destroys ubiquitous rnases . one sample was analyzed for foxp2 expression ; the other sample was stored in a biobank for subsequent validation studies . once collected , samples were stored for a minimum of 48 h at 4c before total rna extraction with the use of the rnaprotect saliva mini kit ( qiagen ) . on - column every attempt was made to adhere to minimum information for quantitative real - time pcr experiments ( miqe ) guidelines established in 2009 to ensure proper and accurate reporting of rt - qpcr data ( bustin et al . relative quantitative gene expression differences of salivary foxp2 were assessed on the applied biosystems quantstudio 7 flex real - time pcr instrument with the use of two reference genes ( gapdh and ywhaz ) previously shown by the maron laboratory to maintain their gene expression across pmas ( maron et al . 2012 ) . inventoried stock sequences of reference and target genes were provided by life technologies : gapdh ( hs03929097 ) , ywhaz ( hs03044281 ) , and foxp2 ( hs00362818_m1 ) . for each salivary sample , foxp2 was run in duplicate , multiplexed one time each with the two reference genes with the path - id multiplex one - step rt - pcr kit ( life technologies ) . to ensure that multiplexing did not interfere with gene amplification , each gene was also run in singleplex one time with the use of a single sample . negative controls with nuclease - free water were run on each plate to ensure that there was no primer the rt - qpcr cycle profile was as follows : reverse transcription , 48c  15 min ; activation of dna polymerase , 95c  10 min ; 40 cycles of pcr , denaturing 95c  15 sec followed by annealing / extension at 60c  1 min . if a sample failed to amplify both reference genes , it was deemed to be of insufficient quality and was not considered in the analysis . the ct method was used for relative gene expression quantification in all samples ( livak and schmittgen 2001 ) . the geometric mean of the ct values of both reference genes were used to calculate cts with the following equation : 
 ct=(meanctfoxp2[(ctgapdhctywhaz ) ] . daily oral feed percentage was extracted from the nursing care notes and calculated across the eight daily feeds for all infants in the study . total number of days required to reach full oral feeding was determined by subtracting pma on the day of the first oral feeding attempt from the pma on the day the nasogastric tube was successfully removed . infants who never required nasogastric tubes to sustain oral feeds were considered to have the developmental maturity of a successful oral feeder from birth . a linear regression model was completed to examine the association between oral feeding and foxp2 gene expression with birth ga , sex , and weight at salivary sample entered as covariates into the model as they are known factors to influence feeding development ( maron et al . 2015 ) . twenty - one premature infants born between 30 and 34 wk ga with a birth weight > 1500 g were recruited for this study . exclusion criteria included major chromosomal anomalies , intraventricular hemorrhage ( > grade ii ) , necrotizing enterocolitis , excessive drug / alcohol use , data missing regarding the date to full oral feeds , and/or the need for prolonged respiratory support ( i.e. , high - flow nasal cannula , continuous positive airway pressure , or ventilator support ) that would have delayed oral feeding trials . saliva samples were taken at the closest approximate time ( within the 3234-wk period ) that an infant commenced oral feeding attempts . there were a few infants ( see those indicated by an   in table 1 ) that attained full oral feeds before the acquisition of the salivary sample . two of these infants were capable of full oral feeds from birth , and the other took 5 d to attain full oral feeds , only briefly requiring nasogastric support before the attainment of a sample . all infants in the tufts medical center nicu are subjectively assessed for oral feeding readiness based on the cue - based feeding algorithm described by ludwig and waitzman ( 2007 ) . salivary samples were collected and processed with previously described techniques in order to simulate routine bedside care of the neonates ( dietz et al . the neonate 's oropharynx was gently suctioned ( < 1 min ) and saliva was immediately stabilized in 500 l of rnaprotect saliva ( qiagen ) . this stabilizing agent halts gene expression changes , inhibits microbial overgrowth , and destroys ubiquitous rnases . one sample was analyzed for foxp2 expression ; the other sample was stored in a biobank for subsequent validation studies . once collected , samples were stored for a minimum of 48 h at 4c before total rna extraction with the use of the rnaprotect saliva mini kit ( qiagen ) . on - column dnase digestion every attempt was made to adhere to minimum information for quantitative real - time pcr experiments ( miqe ) guidelines established in 2009 to ensure proper and accurate reporting of rt - qpcr data ( bustin et al . relative quantitative gene expression differences of salivary foxp2 were assessed on the applied biosystems quantstudio 7 flex real - time pcr instrument with the use of two reference genes ( gapdh and ywhaz ) previously shown by the maron laboratory to maintain their gene expression across pmas ( maron et al . 2012 ) . inventoried stock sequences of reference and target genes were provided by life technologies : gapdh ( hs03929097 ) , ywhaz ( hs03044281 ) , and foxp2 ( hs00362818_m1 ) . for each salivary sample , foxp2 was run in duplicate , multiplexed one time each with the two reference genes with the path - id multiplex one - step rt - pcr kit ( life technologies ) . to ensure that multiplexing did not interfere with gene amplification , each gene was also run in singleplex one time with the use of a single sample . negative controls with nuclease - free water were run on each plate to ensure that there was no primer the rt - qpcr cycle profile was as follows : reverse transcription , 48c  15 min ; activation of dna polymerase , 95c  10 min ; 40 cycles of pcr , denaturing 95c  15 sec followed by annealing / extension at 60c  1 min . if a sample failed to amplify both reference genes , it was deemed to be of insufficient quality and was not considered in the analysis . the ct method was used for relative gene expression quantification in all samples ( livak and schmittgen 2001 ) . the geometric mean of the ct values of both reference genes were used to calculate cts with the following equation : 
 ct=(meanctfoxp2[(ctgapdhctywhaz ) ] . daily oral feed percentage was extracted from the nursing care notes and calculated across the eight daily feeds for all infants in the study . total number of days required to reach full oral feeding was determined by subtracting pma on the day of the first oral feeding attempt from the pma on the day the nasogastric tube was successfully removed . infants who never required nasogastric tubes to sustain oral feeds were considered to have the developmental maturity of a successful oral feeder from birth . these infants achieved a full oral intake of 140 cc / kg / day within the first 7 d of life . a linear regression model was completed to examine the association between oral feeding and foxp2 gene expression with birth ga , sex , and weight at salivary sample entered as covariates into the model as they are known factors to influence feeding development ( maron et al . 2015 ) . the tufts medical center institutional review board approved this study and parental consent was attained prior to the start of the study . we thank all of the parents and babies who participated in this study at the tufts medical center nicu . we acknowledge support from the hearst foundations for the purchase of the quant - studio 7 flex real - time quantitative pcr system . the tufts medical center institutional review board approved this study and parental consent was attained prior to the start of the study . we thank all of the parents and babies who participated in this study at the tufts medical center nicu . we acknowledge support from the hearst foundations for the purchase of the quant - studio 7 flex real - time quantitative pcr system .